CN101528920A

CN101528920A - Multiple mutation variants of serine protease

Info

Publication number: CN101528920A
Application number: CNA2007800389821A
Authority: CN
Inventors: W·埃勒; D·A·埃斯特尔; R·W·J·霍梅斯; B·E·琼斯; M·科尔克曼; C·莱夫朗; 黄浩; A·J·普洛斯; A·肖; W·A·H·范德克莱; L·范马雷维克
Original assignee: Danisco USA Inc
Current assignee: Danisco USA Inc; Danisco US Inc
Priority date: 2006-10-19
Filing date: 2007-08-29
Publication date: 2009-09-09

Abstract

The present invention provides novel Micrococcineae spp serine proteases having multiple substitutions. In particular, the present invention provides serine proteases having multiple substitutions, DNA encoding these proteases, vectors comprising the DNA encoding the proteases, host cells transformed with the vector DNA, and enzymes produced by the host cells. The present invention also provides cleaning compositions (e.g., detergent compositions), animal feed compositions, and textile and leather processing compositions comprising these serine protease variants. In particularly preferred embodiments, the present invention provides mutant (i.e., variant) proteases derived from the wild-type proteases described herein. These variant proteases also find use in numerous applications.

Description

The multiple mutation variants of serine protease

The application is the USSN 11/809 that submitted on May 31st, 2007,104 international application, it is the U.S. Patent Application Serial Number of submitting on October 19th, 2006 co-pending 11/583,334 continuation application, it is a U.S. Patent Application Serial Number 10/576 co-pending, 331 part continuation application, the latter requires the right of priority of the PCT/US2004/039066 of submission on November 19th, 2004, this application requires the right of priority of the present resigned U.S. Provisional Patent Application sequence number 60/523,609 of submission on November 19th, 2003 then.

Technical field

The invention provides new micrococci (Micrococcineae) serine protease that contains multiple replacement.Particularly, the invention provides the serine protease that contains multiple replacement, these proteolytic enzyme of encoding DNA, comprise the DNA of this proteolytic enzyme of encoding carrier, transform the enzyme that host cell that described carrier DNA is arranged and this host cell produce.The present invention also provides cleaning compositions (as detergent composition), animal feedstuff compositions and fabric and the leather processing composition that comprises these serine protease variants.In particularly preferred embodiments, the invention provides mutant (being variant) proteolytic enzyme from wild-type protease described herein.These misfolded proteins enzymes also can be used for multiple application.

Background technology

Serine protease is a subgroup of plurality of enzymes classification, has multiple specificity and biological function (consult as Stroud Sci.Amer., 131:74-88[1974]).Although there is functional diversity in it, the catalysis machine of serine protease is represented by enzyme families different at least two heredity: 1) subtilisin; With 2) and homologous bacterial serine proteolytic enzyme (as trypsinase) relevant with the Mammals Chymotrypsin.These two serine stretch protein enzyme families show highly similar catalyst mechanism (consult as Kraut Ann.Rev.Biochem., 46:331-358[1977]).In addition, although primary structure is uncorrelated, the tertiary structure of these two enzyme families all contains the amino acid conservative property catalysis triplet of being made up of Serine, Histidine and aspartic acid.

On the contrary, the serine protease that subtilisin is relevant with Chymotrypsin all has the catalytic triplet that comprises aspartic acid, Histidine and Serine.In the relevant proteolytic enzyme of subtilisin, these amino acid whose relative orders are aspartic acid-Histidine-Serine from the aminoterminal to the carboxyl terminal.Yet in the relevant proteolytic enzyme of Chymotrypsin, order is Histidine-aspartic acid-Serine relatively.Because the purposes in cleaning and feed applications, many researchs are carried out subtilisin.Other work are conceived in multiple application can be to the functional unsuitable environmental condition that has a negative impact of these enzymes (for example being exposed to oxygenant, sequestrant, extreme temperature and/or pH).However, the active enzyme that still needs to resist these unfavourable condition and reservation in this area or improve this area present known enzyme system is.

Summary of the invention

The invention provides isolating serine protease variants, it has and comprises the aminoacid sequence that at least two amino acid are replaced, and wherein said replacement is carried out on the position of Cellulomonas (Cellulomonas) the 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.The present invention also provides the composition that comprises isolating serine protease variants, described variant contains at least two amino acid to be replaced, and wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described composition comprises at least a variant serine protease, and serine protease has the immunology cross reactivity shown in wherein said variant serine protease and the SEQ ID NO:8.In other embodiment preferred, the sequence of serine protease variants comprises the replacement at least two amino acid positions, and described amino acid position is selected from 1,2, and 3,4,7,8,9,10,11,12,13,14,15,16,18,19,22,24,25,26,27,28,29,30,31,32,33,34,35,36,38,39,40,41,42,43,44,45,46,47,48,49,51,52,54,55,56,57,59,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,96,99,100,101,103,104,105,107,109,110,111,112,113,114,115,116,117,118,119,121,123,124,125,126,127,128,129,130,132,133,134,135,136,137,140,141,142,143,144,145,146,147,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,172,175,176,177,179,180,181,182,183,184,185,186,187,188 and 189, wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.

In some preferred embodiments, described serine protease variants comprises and is selected from least two following replacements: G12D, R14I, R14L, R14M, R14S, R16I, R16L, R16Q, N24E, N24H, N24M, N24T, N24W, R35E, R35F, R35H, T36S, G49A, G54D, G54L, R61V, A64K, G65Q, Q71F, Y75G, S76A, S76L, S76N, S76T, S76V, R79K, R79T, Q81K, Q81P, T86K, A93G, A93H, A93S, S99A, T109M, N112E, T116E, R123F, R123L, R123Q, R123S, R127A, R127K, R127Q, R159E, R159F, R159G, R159K, R159L, R159Q, R179N, R179Q, I181K, I181Q, I181T, D184N, D184T and S187Q, wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.In other embodiment preferred, described serine protease variants comprises and is selected from R16Q/R35F/R159Q, R16Q/R123L, R14L/R127Q/R159Q, R14L/R179Q, R123L/R127Q/R179Q, R16Q/R79T/R127Q, R16Q/R79T, the multiple replacement of R35E/R123L/R127Q/R179Q and G12D/R35E/G63R/R79K/T109M is carried out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiment preferred, described serine protease variants comprises following replacement: R123L, R127Q and R179Q carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

In other embodiment preferred, described serine protease variants comprises and is selected from least two following replacements: D2G, D2Q, V3I, V3L, N7A, N7L, N7S, I11A, I11Q, R14E, N24A, N24E, N24H, N24L, N24M, N24Q, N24T, N24V, T36D, T36F, T36G, T36H, T36I, T36L, T36N, T36P, T36R, T36S, T36V, T36W, T36Y, A38D, A38F, A38H, A38L, A38N, A38R, A38S, G49F, S51A, G54A, G54D, G54H, G54K, G54L, G54M, G54R, N55F, A64H, A64N, A64R, A64W, A64Y, G65L, G65P, G65Q, G65R, G65S, G65T, G65Y, G65V, V66A, V66D, V66E, V66H, V66I, V66L, N67A, N67G, N67L, N67K, L69H, L69S, L69V, A70D, A70H, A70S, Q71A, Q71G, Q71H, Q71I, Q71K, Q71M, Q71N, N73S, N73T, N74G, Y75F, Y75G, Y75I, S76L, S76Y, S76V, S76W, G77S, G77T, G78A, G78D, G78H, G78N, G78S, G78T, R79P, V80H, V80L, Q81H, Q81K, Q81V, H85Q, H85T, V90I, V90P, V90S, S92G, W103I, W103M, H104K, T109A, T109H, T109I, S114G, T116F, P118A, P118F, P118H, P118R, E119K, E119R, N145E, N145I, N145Q, V150L, R159F, N170Y, G177M, R179A, R179D, R179E, R179I, R179K, R179L, R179M, R179N, R179T, R179Y, R179V, I181H, T183I, G186E, G186I, G186V, S187P, S187T and S188M carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

In other embodiments, described serine protease variants comprises and is selected from least two following replacements: F1A, F1T, D2A, D2H, D2N, V3T, N7H, N7I, A8G, A8K, T10G, T10K, I11S, I11T, G12W, G13M, S15F, N24F, N24S, A30S, R35F, T36C, A38G, A38I, A38K, A38V, A38Y, T40S, T40V, A41N, N42H, F47I, F47M, G49A, G49K, G49L, S51F, S51Q, G54I, G54Q, N55K, N55Q, R61M, T62I, G63Q, G63V, G63W, A64F, A64I, A64K, A64L, A64M, A64Q, A64S, A64T, A64V, G65A, G65H, V66M, V66N, N67D, N67F, N67H, N67Q, N67R, N67S, N67T, N67V, N67Y, L68W, L69W, A70G, Q71D, Q71F, Q71L, Q71R, V72I, N73H, S76E, S76I, S76K, S76A, S76N, S76Q, S76R, S76T, G77N, G77Y, G78I, S78R, G78V, R79G, V80F, Q81D, Q81I, A83N, H85R, H85K, H85L, T86A, P89N, V90A, V90L, V90T, T107H, T107M, T107S, T107V, T109G, T109L, T109P, T109R, A110S, A110T, N112I, P118E, P118I, P118K, P118Q, R123E, R123I, I126L, R127F, I28L, T129S, E133Q, L142V, A143N, A143S, N145G, N145L, N145T, V150M, T151L, R159E, T163L, Q167N, N170A, N170D, N170L, 171S, G177S, I181G, I181N, T182V, T183K, T183M, D184F, D184H, D184Q, D184R, S185I, S185V, S187E and S187L carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

In other embodiments, described serine protease variants comprises and is selected from least two following replacements: D2P, A8G, T10C, T10L, I11E, I11Q, I11T, I11W, G12D, G12I, G12N, G12Q, G12S, G12V, R14A, R14C, R14E, R14D, R14G, R14I, R14N, R14Q, R14S, R14T, S15C, S15E, S15H, S15R, S15Y, R16C, R16D, R16E, R16T, R16V, A22C, A22S, N24E, R35A, R35C, R35D, R35E, R35H, R35M, R35N, R35P, R35Q, R35S, R35T, R35V, T36C, A38C, A38D, A41C, A41D, T44E, T46C, T46E, T46F, T46V, T46Y, F47R, A48E, G49A, G49C, G49E, G49H, G49L, G49N, G49Q, G49V, G54C, N55G, D56L, Y57G, F59W, R61E, R61M, R61T, R61V, G91Q, S99A, T100A, T100R, T107R, T109E, N112P, S113C, S114C, P118K, P118R, E119G, E119R, E119T, E119V, E119Y, T121E, T121F, T121L, R123C, R123D, R123E, R123F, R123H, R123N, R123Q, R123S, R123T, R123V, R123W, R123Y, G124D, L125Q, R127D, R127E, R127K, R127Q, R127S, P134R, T151C, T151L, S155C, S155I, S155W, S155Y, R159D, R159E, R159Q, R159S, R159T, R159V, T163D, F165E, F165W, Q167E, N170C, N170D, G177D, R179D, R179E and M180L carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, described serine protease variants comprises and is selected from least two following replacements: F1N, F1P, D2I, D2M, D2T, D2V, A8R, A8T, T10D, T10E, T10F, T10M, T10Q, T10Y, G12H, G12P, G12Y, G13D, G13E, R14H, R14L, R14M, S15F, S15G, S15N, R16I, R16Q, N24G, N24T, I28V, R35K, T36V, A41S, A41T, N42D, T44C, F47V, G49F, G49K, G49S, S51A, S51C, S51L, S15M, G54E, N55A, D56F, R61K, R61Q, A64C, G65D, V66N, A70G, A70M, A70P, R79T, R79V, Q81A, Q81G, Q81P, A83E, A83D, A83H, T86E, A87C, A87E, A88F, S92T, S99G, S99H, S99K, S99Q, T100K, T100Q, W103L, T109K, N112D, N112E, S113A, S113D, S114E, T115C, T116G, T116N, P118A, P118C, P118G, P118W, E119A, E119L, E119N, E119Q, E119S, T121A, T121D, R123A, R123G, R123I, R123K, R123M, A132S, L125M, R127A, R127C, T128A, S140P, L141M, T151V, S155E, S155F, S155T, S155V, N157D, R159A, R159C, R159K, R159M, R159N, T160D, T163C, F165H, N170L, I172A, Q174C, Q174S, Q174T, A175T, G177E, R179C, R179F, R179I, R179L, R179M, R179N, R179S, R179T, R179V, R179W, R179Y, S187E and S188E carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

In other embodiments, the invention provides serine protease variants, wherein the aminoacid sequence of this proteolytic enzyme comprises and is selected from following at least two amino acid and replaces: V3R, I4D, I4G, I4P, Y9E, Y9P, T10F, T10W, T10Y, G12D, S18E, A22C, A22S, A22T, N24T, G26E, G26I, G26K, G26Q, G26V, G26W, F27V, F27W, I28P, I28T, T29E, A30M, A30N, A30P, A30Y, G31H, G31M, G31N, G31V, G31Y, C33E, C33L, C33M, C33N, A38D, A38G, T39R, T40D, T40H, T40N, T40P, T40Q, R43D, P43G, P43H, P43K, P43L, P43N, G45A, G45V, T46V, T46Y, T46W, A48P, Y57M, Y57N, F59K, T62G, T62R, A70G, A70P, N73P, R79T, Q81A, Q81D, Q81F, Q81G, Q81H, Q81P, Q81S, A83H, G84C, G84P, P89W, G91L, A93S, R96C, R96E, R96F, S99A, T100A, C105E, C105G, C105K, C105M, C105N, C105P, C105S, C105W, T121E, R123F, R123N, R123W, R123Y, L125A, T128A, T128C, T128G, T128S, T128V, I28W, T129W, S137R, S140P, Q146P, A147E, S155F, S155K, S155P, S155R, S155W, S155Y, G156I, G156L, G156P, C158G, C158H, C158M, R159K, T160I, G161I, G161L, G161V, T164G, T164L, F166S, Q167L, P168Y, Y176P, G186S and S188A, wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.In some embodiments, comprise in the aminoacid sequence of this serine protease variants and be selected from least two following replacements: A8G, T10C, T10L, G12A, G12H, S15C, S15N, S15Q, S15R, S15T, N24E, N24S, G25S, F27I, G31A, H32A, C33D, T36V, T39V, A41S, T46F, G49A, S51V, F59W, Q71A, Q71Y, N74F, R79V, Q81C, Q81E, A83E, A83F, A83M, A83R, G84M, G84V, T86I, T86M, T86S, A87E, A87S, P89A, V90A, V90M, S92T, A93D, S99G, T100Q, T101S, W103N, C105A, C105L, C105T, C105Y, T107A, T107F, T107L, T107Q, T107S, T110D, A110G, L111K, V115I, V115L, T116Q, Y117K, Y117Q, Y117R, Y117V, P118T, E119L, T121A, T121D, R123I, R123K, R123L, R123Q, R123T, L125M, R127F, R127K, R127Q, T129Y, V130T, A132C, P134W, L141C, A143H, G144A, V150N, T151C, G153K, G153V, G154L, G154R, S155T, R159Q, R159T, R159V, T160E, T160Q, G161K, G162P, T163I, F166A, F166C, P168I, N170D, N170E, G177N, R179K, M180L, T182L, T183A, T183I, T183P, S185R, G186P, S188C, S188E, S188G, S188M, S188T, S188V and P189S carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQID NO:8.

The present invention also provides serine protease variants, and the aminoacid sequence of wherein said proteolytic enzyme comprises and is selected from least two following replacements: F1T, T10N, R14D, R14G, R14I, R14L, R14N, R14Q, R14T, N24A, N24E, N24H, N24L, N24Q, N24T, N24V, R35A, R35E, R35F, R35L, R35Q, R35T, T36G, T36I, T36N, T36S, A38D, A38F, A38H, A38N, A38R, G49A, G49S, S51D, G54D, G54E, N55E, N55F, A64I, G65D, G65P, G65Q, G65S, G65T, G65V, N67D, L69S, N73T, N74G, Y75F, Y75G, S76D, S76E, S76I, S76L, S76N, S76T, S76V, S76Y, G77T, G78A, G78D, R79A, R79D, R79E, R79G, R79L, R79M, R79P, R79S, R79T, R79V, Q81E, A83E, H85Q, H85T, T86D, T86E, V90I, V90P, V90S, V90T, S99N, S99V, T107E, T107H, T107S, T107V, T109E, N112D, N112E, N112L, N112Q, N112V, T116E, T116Q, T121E, R123A, R123D, R123E, R123F, R123H, R123I, R123L, R123N, R123Q, R127A, R127Q, T129S, L142V, N145E, R159D, R159E, R159F, R159N, R159Q, N170D, N170Y, I172T, R179A, R179D, R179E, R179I, R179K, R179M, R179N, R179T, R179Y, R179Y, I181L and G186N carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some alternative embodiment, the aminoacid sequence of this proteolytic enzyme comprises and is selected from least two following replacements: F1D, V3L, N7L, A8E, A8G, T10D, T10E, G12D, G13S, R14A, R14K, R14S, R14M, S15W, I19V, N24M, R35H, R35M, R35S, R35W, R35Y, T36D, T36H, A38L, A38S, A38T, A38Y, T40V, A41D, A41N, A48E, G49F, G49H, S51H, S51Q, S51T, S51V, R61E, R61H, R61M, R61S, R61T, G63D, A64F, A64H, A64L, A64M, A64N, A64P, A64Q, A64S, A64T, A64V, A64W, A64Y, G65L, G65Y, N67E, N67G, N67H, N67S, N67T, A70D, A70G, A70H, Q71D, Q71G, Q71H, Q71S, V72I, S76Q, S76W, G77S, Q81D, Q81H, Q81V, H85L, H85M, V90N, S92A, S92G, A93D, A93E, A93S, S99D, S99T, T101S, W103M, T107A, T107I, T107M, T107N, T109A, T109G, T109I, A110S, N112Y, S113T, S114A, V115A, T116F, T121D, N121I, R123G, R123S, R123T, R123V, R123Y, R127H, R127K, R127E, R127S, R127Y, N145D, N145T, R159A, R159C, R159K, R159L, R159S, R159Y, T160E, T163D, N170L, R179L, T182V, T183E, T183I and S185N carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, described serine protease variants comprises and is selected from least two following replacements: D2Q, V3L, N7L, I11A, I11Q, R14I, R14M, R16L, R16Q, N24A, N24E, N24H, N24M, N24Q, N24T, N24V, R35F, R35L, T36D, T36G, T36H, T36I, T36L, T36N, T36P, T36S, T36W, T36Y, A38L, A38R, A38S, A48Q, G49A, G54D, G54I, G54Q, G54N, R61V, A64F, A64H, A64Y, G65L, G65P, G65Q, G65S, G65T, G65Y, V66H, N67A, N67G, N67L, N67S, N67V, N67Y, L69H, L69S, Q71I, N73T, N74G, Y75F, Y75G, Y75I, S76A, S76D, S76E, S76I, S76L, S76N, S76T, S76V, S76W, S76Y, G77T, G78D, R79G, R79P, Q81P, H85F, H85K, H85L, H85Q, H85R, P89D, S92A, A93T, A93S, S99A, S99D, S99N, S99T, S99W, T109E, N112E, S113A, S114G, T116F, T121D, R123F, R123I, R123L, R127A, R127F, R127G, R127H, R127K, R127L, R127Q, R127S, R127T, R127Y, A132V, P134E, A143N, N157D, R159D, R159E, R159F, R159H, R159K, R159N, R159Y, G161K, N170Y, R179V, I181Q, D184F, D184H, G186E, G186I, G186V and S187P carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQID NO:8.

In other embodiments, comprise in the aminoacid sequence of this serine protease variants and be selected from least two following replacements: F1T, A8D, A8G, T10E, T10L, T10Q, I11L, I11S, I11T, G12D, G12Y, R14E, R14L, R14N, R14P, S15E, R16A, R16G, R16I, R16N, N24L, N24S, V31F, R35A, A38D, A38F, A38N, A38V, A38Y, T40V, A41N, N42H, G49F, G49H, G49S, S51Q, S51T, G54A, G54L, G54M, N55F, R61H, R61K, R61M, R61S, R61T, A64N, A64S, A64T, A64V, A64W, G65R, G65V, V66D, N67F, N67K, N67M, N67Q, N67T, L69W, A70G, A70P, Q71D, Q71F, Q71H, Q71L, Q71T, G77N, G77S, G78A, G78N, R79D, V80H, V80L, H85T, H85Y, T86N, A88F, P89N, P89V, V90I, V90P, V90T S92G, A93D, A93E, S99G, L111D, L111E, N112D, N112G, N112L, N112Q, S113G, T121E, R123E, R123K, R123Q, L125V, P134G, S140A, L142V, A143S, N145D, V150L, R159A, R159C, R159L, R159V, T160E, G161E, T163D, T163I, N170D, N170L, R179D, R179E, R179K, R179N, R179T, I181H, T183I, D184R, D184L, D184Q, D184T, S185W, S185I, G186L, S187E, S187Q and S188Q carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, comprise in the aminoacid sequence of this serine protease variants and be selected from least two following replacements: A8R, A8S, A8T, A8V, R14E, R14L, R14M, R16Q, N24A, N24E, N24Q, N24T, R35F, R35L, T36D, T36G, T36I, T36N, T36P, T36S, A38D, A38F, A38L, A38R, A38S, S51A, S51D, G54D, G54I, N55E, N55F, N55S, R61M, R61T, G63V, A64H, A64N, A64S, G65Q, G65P, G65R, G65S, G65T, G65Y, V66D, N67D, S76E, N67F, N67G, N67L, N67M, N67S, N67T, N67V, N67Y, L69H, L69S, L69V, L69W, N73T, N74G, Y75F, Y75G, S76C, S76D, S76I, S76L, S76N, S76W, S76Y, S76V, G77T, G78D, R79C, R79D, R79E, R79G, R79P, Q81V, A83N, T85A, H85Q, T86F, T86I, T86L, V90I, V90N, V90P, V90S, V90T, A93D, A93E, T107M, T107N, T107S, T109A, T109E, T109I, N112E, T121D, T121E, R123D, R123E, R123F, R123I, I126L, R127A, R127H, R127K, R127L, R127Q, R127S, R127Y, P134A, P134E, L142V, A143N, N145E, N145S, R150Y, R159C, R159D, R159E, R159F, R159K, R159Q, G161E, T163D, N170Y, I172V, G177M, R179A, R179D, R179E, R179I, R179K, R179L, R179M, R179N, R179T, R179V, R179Y, M180D, T182V, T183I, G186E, G186V and S187P carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, the aminoacid sequence of described serine protease variants comprises and is selected from least two following replacements: V3L, I4M, A8E, A8H, A8L, A8N, A8P, I11T, R14I, R14Q, R16L, N24H, N24L, N24M, N24V, R35A, R35E, T36V, T36Y, A38H, A38I, A38N, T40V, A41N, G49A, G49L, S51F, S51Q, G54A, G54E, G54M, G54Q, N55Q, N55V, R61V, T62I, G63D, G63L, G63P, G63Q, A64F, A64I, A64L, A64M, A64Q, A64R, A64T, A64V, A64W, G65A, G65D, V66E, N67A, N67C, N67Q, N67R, L69Q, A70G, A70P, A70S, Q71D, Q71M, S76A, S76Q, S76T, G77N, G77Q, R79L, Q81E, Q81H, Q81I, A83D, A83I, H85L, H85R, T86E, T86M, A88F, V90L, S92C, S92G, A93Q, R96K, T101S, W103M, W103Y, T107A, T107E, T107H, T107Q, T107V, T109G, T109H, T109L, T109N, A110S, A110T, N112D, S114G, T116F, T121L, R123A, R123H, R123K, R123L, R123P, R123Q, L125V, R127F, R127T, T129G, T129S, A132V, P134D, P134G, S140A, N145G, N145P, N145Q, N145T, Q146D, T151V, R159A, R159H, R159L, R159N, R159V, S161K, F166Y, N170C, N170D, P171M, A175T, A175V, Y176L, R179W, T182W, T183E, T183K, T183L, T183Q, G186I, G186L, G186P, G186T, S187E, S187T and S188E carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, the aminoacid sequence of described serine protease variants comprises and is selected from least two following replacements: T10A, T10G, T10L, I11A, I11S, I11T, G12I, R14G, R14M, S15E, S15F, S15G, R16K, R16N, A22V, N24A, N24E, N24L, N24Q, N24T, N24V, G34A, T36G, T36I, T36N, T36S, A38F, A38T, G49A, G49F, S51A, G65V, L69H, L69S, Q71I, N73T, N74G, S76D, S76L, S76V, S76W, S76Y, G77T, V80A, V90I, V90P, S99N, S99V, T107K, T107R, N112S, S118A, E119R, R127F, P134D, P134E, P134H, P134L, P134R, P134V, S140A, L142V, V150L, 159F, R159K, T163I, F166Y, Q167N, N170Y, R179V, T182V, G186E, G186S and G186V carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQID NO:8.

The present invention also provides the serine protease variants of comparing the stability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some embodiments, described variant is compared the thermostability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some particularly preferred embodiments, described variant comprises and is selected from following multiple replacement: T121E/R123F/R159E,

R79T/R127Q/R179Q，R16Q/R79T/R127Q，

R16Q/R79T/R123L/R159Q/R179Q，R16Q/R79T/R123L，R16Q/R79T，

R16Q/R123L/R159Q，R14Q/T121E，R14L/R79T，R123L/R159Q，

R123L/R127Q/R159Q，G12D/S15E/R35D/R123F/R159E，

G12D/S15E/R159E，G12D/S15E，G12D/R35H/T121E/R123Q，

G12D/R35H/R123Q，G12D/R35H/R123F/R159E，G12D/R35H，

G12D/R35E/R123Q，G12D/R35E，G12D/R159E，G12D/R14Q/S15E/R35D，

G12D/R14Q/R35H，G12D/R14Q/R159E，G12D/R14E，G12D/R127Q/R159E，

G12D/R123E/R159E and R35E/R123L/R127Q/R175Q carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some alternative embodiment, described variant is compared the LAS stability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some particularly preferred embodiments, described variant comprises and is selected from following multiple replacement: T121E/R123F/R159E, S15E/T121E/R123Q, and S15E/R35H/R159E,

S15E/R35E/R159E，S15E/R35E/R127Q/R159E，S15E/R35E，

S15E/R35D/T121E/R123Q，S15E/R35D/R123Q，S15E/R35D/R123F/R159E，

S15E/R35D，S15E/R159E，S15E/R127Q，S15E/R123Q，S15E/R123E，

R79T/R127Q/R179Q，R35H/R159E，R35H/R127Q/R159E，

R35H/R123D/R159E，R35F/R61S/R159Q，R35F/R159Q，

R35E/T121E/R123E，R35E/R159E，R35E/R127Q，R35D/R159E，

R35D/R127Q/R159E，R35D/R127Q，R35D/R123Q/R159E，

R16Q/R79T/R159Q/R179Q，R16Q/R79T/R127Q，

R16Q/R79T/R123L/R159Q/R179Q，R16Q/R79T/R123L/R159Q，

R16Q/R79T/R123L，R16Q/R79T，R16Q/R61S/R159Q/R179Q，

R16Q/R61S/R123L/R159Q，R16Q/R35F/R61S/R159Q，R16Q/R35F/R159Q，

R16Q/R35F/R123L/R159Q，R16Q/R35F，R16Q/R159Q/R179Q，

R16Q/R159Q，R16Q/R127Q/R179Q，R16Q/R127Q/R159Q，

R16Q/R123L/R159Q，R14Q/T121E，R14Q/R35E/T121E，

R14Q/R35E/R159E，R14Q/R35E，R14Q/R35D/R127Q，

R14Q/R35D/R123E/R159E，R14Q/R35D/R123D/R159E，R14Q/R35D，

R14Q/R123Q，R14L/R79T/R127Q/R159Q，R14L/R79T，

R14L/R61S/R79T/R123L，R14L/R61S/R123L，

R14L/R35F/R79T/R123L/R159Q，R14L/R35F/R61S，

R14L/R127Q/R159Q/R179Q，R14L/R123L/R159Q，

R14I/R35E/T121E/R159E，R14I/R35E/R127Q，R14I/R35E/R123E，

R14I/R35D/R159E，R14I/R35D/R127Q/R159E，R14E/S15E/R35H，

R14E/R35H/R127Q，R14D/S15E/R35E/R159E，R14D/R35H/R123Q/R159E，

R127Q/R159E，R127Q/R159Q，R123Q/R159E，R123Q/R127Q/R159E，

R123L/R159Q，R123L/R127Q/R159Q，R123F/R159E，

R123E/R127Q/R159E，R123E/R127Q，G12D/S15E/R35H/R159E，

G12D/S15E/R35H/R123F/R127Q/R159E，G12D/S15E/R35E/R159E，

G12D/S15E/R35D/R127Q，G12D/S15E/R35D/R123F/R159E，

G12D/S15E/R35D/R123E，G12D/S15E/R35D，G12D/S15E/R159E，

G12D/S15E，G12D/R35H/T121E/R123Q，G12D/R35H/R159E，

G12D/R35H/R123Q/R159E，G12D/R35H/R123Q，

G12D/R35H/R123F/R159E，G12D/R35H，G12D/R35E/R159E，

G12D/R35E/R123Q/R159E，G12D/R35E/R123Q，G12D/R35E，

G12D/R35D/R159E，G12D/R35D/R127Q，G12D/R35D/R123Q/R159E，

G12D/R35D，G12D/R159E，G12D/R14Q/S15E/R35D，G12D/R14Q/R35H，

G12D/R14Q/R35E/R127Q/R159E，G12D/R14Q/R35D/R123H，

G12D/R14Q/R159E，G12D/R14I/R35H，G12D/R14E，

G12D/R14D/R35H/R123D/R127Q，G12D/R127Q/R159E，

G12D/R123E/R159E，R127A/R159K，R14I/G65Q，

R14I/G65Q/N67L/R159K，R14I/G65Q/N67L/Y75G/R127A/R159K，

R14I/G65Q/R159K，R14I/G65Q/S76V/R127A/R159K，R14I/R127A，

R14I/R127A/R159K，R14I/R159K，R14I/R35F，R14I/R35F/G65Q，

R14I/R35F/G65Q/R127A/R159K，R14I/R35F/N67L/R127A/R159K，

R14I/R35F/R127A/R159K，R14I/R35F/R159K，R14I/S76V，

R14I/T36S/G65Q/R127A/R159K，R35F/R127A/R159K，R35F/S76A/R127A，

024A/G049A/A093H/S099N/R127K/A143N/R159K/I181Q，

N024A/S076A/A093H/S099G/R127K/R159K，

N024A/S076T/A093S/S099G/R127K/R159K，

N024E/G049A/A093G/S099G/R127K/A143N/R159K/I181T，

N024E/G049A/A093H//R127K/A143N/R159K/I181Q，

N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I，

N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q，

N024H/G049A/A093T/S099A/R127K/A143N/R159K/I181Q，

N024H/S076A/A093G/S099G/R127K/R159K，

N024H/S076A/A093H/S099G/R127K/R159K，

N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H，

N024H/S076A/A093T/S099G/R127K/R159K，

N024H/S076N/A093Q/S099W/R127K/R159K，

N024H/S076V/A093Q/S099G/R127K/R159K，

N024L/G049A/A093H/S099A/R127K/A143N/R159K/I181Q，

N024L/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N024L/S076V/A093H/S099G/R127K，

N024L/S076V/A093S/S099A/R127K/R159K，

N024M/G049A/A093G/S099A/R127K/A143N/R159K/I181Q，

N024M/G049A/A093H/S099D/R127K/A143N/R159K/I181Q，

N024M/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N024M/G049A/A093S/S099W/R127K/A143N/R159K/I181Q，

N024Q/G049A/A093H/S099A/R127K/A143N/R159K/I181T，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N024Q/S076A/A093H/S099A/R127K/R159K/T039N，

N024Q/S076A/A093H/S099W/R127K/R159K，

N024Q/S076I/A093T/S099G/R159K，N024Q/S076T/A093S/R127K/R159K，

N024S/S076A/A093G/S099G/R127K/R159K/G054A，

N024S/S076A/A093H/S099T/R127K/R159K，

N024S/S076A/A093S/S099W/R127K/R159K，

N024S/S076A/A093T/S099W/R127K，

N024S/S076T/A093Q/S099W/R127K/R159K，

N024S/S076Y/A093T/S099A/R127K/R159K，

N024T/G049A/A093G/S099A/R127K/A143N/R159K/I181Q，

N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181Q，

N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181T，

N024T/G049A/A093H/S099D/R127K/A143N/R159K/I181Q，

N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N024T/G049A/A093T/S099A/R127K/A143N/R159K/I181T，

N024T/S076N/A093Q/S099T/R127K/R159K，

N024T/S076T/A093T/S099N/R127K/R159K，N024V/S076A/R127K/R159K，

N024V/S076V/A093Q/S099G/R127K/R159K，

N024W/A093G/S099W/R127K/R159K，

N024W/G049A/A093H/S099A/R127K/A143N/R159K/I181Q，

N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181K，

N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N024W/S076A/A093T/S099A/R127K/R159K，

N024W/S076I/A093Q/S099G/R127K/R159K，

N024W/S076N/A093T/S099G/R159K，

N024W/S076T/A093H/S099A/R127K/R159K，

N024W/S076T/A093H/S099W/R127K/R159K，

N024W/S076V/A093H/S099A/R127K/R159K，

N024W/S099W/R127K/R159K，N24A/G54E/S76D/A93G/R127K/R159K，

N24E/A93G/R127K/R159K，N24E/G54L/S76E/A93G/R127K/R159K，

N24E/G54Q/A93S/R127K/R159K，N24E/S76D/A93T/R127K/R159K，

N24H/A93H/R127K/R159K，N24H/G54E/A93G/R127K/R159K，

N24H/S76D/A93H/R127K/R159K，N24L/A93G/R127K/R159K，

N24L/G54E/A93G/R127K/R159K，N24L/G54L/A93G/R127K/R159K，

N24L/G54Q/S76A/A93H/R127K/R159K，N24L/S76T/A93G/R127K/R159K，

N24L/S76T/A93H/R127K/R159K，N24M/A93G/R127K/R159K，

N24M/A93H/R127K/R159K，N24M/A93S/R127K/R159K，

N24M/A93T/R127K/R159K，N24M/G54E/A93H/R127K/R159K，

N24M/G54E/S76N/A93S/R127K/R159K，

N24M/G54I/A93H/R127K/R159K/S187I，N24Q/A93G/R127K/R159K，

N24Q/G54D/A93H/R127K/R159K，N24Q/G54I/A93G/R127K/R159K，

N24Q/G54I/S76E/A93H/R127K/R159K，N24Q/G54Q/A93G/R127K/R159K，

N24Q/G54Q/S76T/A93H/R127K/R159K，N24Q/S76A/A93G/R127K/R159K，

N24T/G54D/S76V/A93G/R127K/R159K，

N24T/G54E/S76V/A93H/R127K/R159K，N24T/G54I/A93G/R127K/R159K，

N24T/G54N/A93H/R127K/R159K，N24T/G54Q/S76N/A93G/R127K/R159K，

N24T/G54Q/S76V/R127K/R159K，N24T/S76I/R127K/R159K，

N24T/S76L/A93G/R127K/R159K，N24W/A93G/R127K/R159K，

N24W/G54D/A93H/R127K/R159K，

N24W/G54I/S76A/A93H/R127K/R159K，

N24W/S76A/A93H/R127K/R159K，N24W/S76E/A93G/R127K/R159K，

R014I/S076A/A093G/R127K/R159K/I181T，

R014I/S076A/A093H/R127K/R159K/I181K，

R014I/S076A/A093H/R127K/R159K/I181Q，

R014I/S076A/A093H/R127K/R159K/I181T，

R014I/S076D/A093H/R127K/R159K/I181Q，

R014I/S076D/A093S/R127K/R159K/I181T，

R014I/S076E/A093S/R127K/R159K/I181Q，

R014I/S076E/A093T/R127K/R159K/I181K，

R014I/S076I/A093S/R127K/R159K/I181Q，

R014I/S076N/A093H/R127K/R159K/I181Q，

R014I/S076T/A093G/R127K/R159K/I181Q，

R014K/S076A/A093G/R127K/R159K/I181K，

R014K/S076E/A093H/R127K/R159K/I181K，

R014K/S076T/A093H/R127K/R159K/I181Q，

R014L/S076A/A093H/R127K/R159K，

R014L/S076A/A093H/R127K/R159K/I181Q，

R014L/S076D/A093H/R127K/R159K/I181T，

R014L/S076E/A093H/R127K/R159K/I181K，

R014M/S076A/A093G/R127K/R159K/I181K，

R014M/S076A/A093G/R127K/R159K/I181T，

R014M/S076A/A093H/R127K/R159K/I181T，

R014M/S076A/A093S/R127K/R159K/I181K，

R014M/S076A/A093S/R127K/R159K/I181T，

R014M/S076A/A093T/R127K/R159K/I181Q，

R014M/S076D/A093S/R127K/R159K/I181T，

R014M/S076E/A093G/R127K/R159K/I181T，

R014M/S076E/A093H/R127K/R159K/I181T，

R014M/S076E/A093S/R127K/R159K/I181T，

R014M/S076N/A093G/R127K/R159K/I181K，

R014M/S076N/A093G/R127K/R159K/I181T，

R014M/S076N/A093H/R127K/R159K/I181Q，

R014M/S076N/A093H/R127K/R159K/I181T，

R014M/S076N/A093S/R127K/R159K/I181T，

R014M/S076N/A093T/R127K/R159K/I181T，

R014M/S076T/A093H/R127K/R159K/I181K，

R014M/S076V/A093G/R127K/R159K/I181Q，

R014M/S076V/A093H/R127K/R159K/I181Q，G54E/R14L，G54L/R127S，

N24D/G54F/R127C，N24E/R127S，N24E/R159C，N24G/G54I/R127S，

N24H/R159Y/T46I，N24I/R127V/R14V，N24T/R127Q/R179F，

R127A/R159V/R179F，R127C/R14W，R127S/R159N/R123L，

R14A/N24F/R159L，R14A/R127L，R14A/R127Y/R159W，R14A/R159W，

R14C/S114F/R159G，R14F/R127L/R159F，R14F/R127Q/R159W，

R14F/R127S/R159V，R14F/R127V/R159F，R14G/N24L/R159G，

R14G/N24S/R127C，R14G/R127C/G63E，R14G/R127G，R14G/R127P，

R14L/N24S/R159F，R14L/N24V/R127S/R159I，R14L/R123L，

R14L/R127C/R159G，R14L/R127S，R14L/R127S/R159G，R14L/R127V，

R14L/R127V/R159F，R14L/R127W/R123Y，R14L/R127Y，

R14L/R127Y/R159F，R14L/R159G，R14L/R159L，R14L/R159S，

R14L/R159V，R14L/R159W，R14M/N24L/R159S/R123V，R14M/R159F，

R14Q/R123F，R14S/N24E/R127W，R14S/N24L/R159G，R14S/R127L/R159F，

R14S/R127V，R14T/R14P/R159F，R14T/N24A，R14T/N24T/R127Q，

R14T/N24T/R127Y/R159W，R14T/R127Y，R14V/N24A/R127I/R159A，

R14V/N24D/R127C，R14V/N24G/P189S，R14V/N24S，

R14V/N24Y/R127S/R159G，R14V/R127A，R14V/R127C/R159S，

R14V/R127M/R159V，R14V/R127S/R159G，R14V/R127T/R159Y，

R14V/R127V，R14V/R159F，R14V/R159V，R14V/R159W，

R14W/N24T/R123E，R14W/R123L，R14W/R123V，R14W/R127Q/R159W，

R14W/R159V，R159V/G49D，G012D/R035E/G065E，G012D/R035E/G065E，

G012D/R035E/Q081P，G012D/R035E/R016S/A064T，G012D/R035E/R159W，

G012D/R035E/R179I，G012D/R035E/S092T/I181V，

R14I/N24A/A64K/R123F/R159E/D184T，R14I/A64K/R123F/R159F/D184T，

R14I/A64K/R123F/R159E/D184T，R14I/N24Q/R35E/A64K/R123F/D184T，

R14I/N24Q/A64K/N67S/R123F/R159F/D184T，

R14I/N24A/R35E/A64K/N67S/R123F/R159E/D184T，

R14I/N24A/R35E/A64K/N67S/G78D/R123F/D184T，

R14I/N24A/R35D/A64K/G78D/R123F/R127K/R159E/D184T，

R14I/N24A/R35D/A64K/R123F/R127K/R159F/D184T，

R14I/N24T/R35D/A64K/G78D/R123F/R127Q/R159F/D184T，

R14I/A64K/R123F/D184T，R14I/N24A/A64K/R123F/R159N/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，

R14I/N24E/R35E/A64K/G78D/R123F/R127Q/R159F/D184T，

R14I/N24E/A64K/R123F/R127K/R159K/D184T，

R14I/N24A/A64K/R123F/D184T，R14I/A64K/R123F/R127K/R159F/D184T，

R14I/A64K/R123F/R159E/D184T，R14I/A64K/R123F/R159N/D184T，

R14I/A64K/R123F/R159K/D184T，

R14I/A64K/R123F/R127Y/R159E/D184T，

R14I/N24A/R35E/A64K/N67A/G78D/R123F/D184T，

R14I/A64K/R123F/R127Y/R159K/D184T，

R14I/N24Q/A64K/R123F/R127Q/R159K/D184T，

R14I/A64K/R123F/R159K/D184T，

R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T，

R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T，

R14I/N24A/R35E/A64K/G78D/R123F/R127K/R159E/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，

R14I/N24A/R35D/A64K/N67A/R123F/R159F/D184T，

R14I/N24E/R35E/G54D/A64K/N67L/G78D/R123F/R127K/D184T，

R14I/A64K/G78D/R123F/R127Q/R159N/D184T，

R14I/N24A/R35E/A64K/G78D/R123F/R159N/D184T，

R14I/A64K/R123F/R127K/R159E/D184T，

R14I/N24A/R35E/A64K/N67S/G78D/R123F/R127K/R159F/D184T，

R14I/A64K/G78D/R123F/R159E/D184T，

R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T，

N24T/R35D/G78D/R159K，N24T/R35E/N67A/G78D/R127Q，N24Q/R35E，

R127K/R159N，R35D/R159E，R35E/G54D/N67S/G78D/R159K，

N24Q/G54D/G78D/R159N，R127K/R159E，R127Q/R159K，

N24E/R35E/G54D/N67S/R127K/R159N，R35D/G78D/R159K，N67S/R159E，

G54D/R127K/R159K，G78D/R127K/R159K，G78D/R127K/R159E，

N24E/R35D/G78D/R127K/R159N，R35D/G78D/R127K/R159N，

N24A/R35E/G78D/R159N，N24Q/R35D/N67S/R127K/R159E，

N24T/R35D/G78D/R159K，N67S/G78D/R127K/R159K，

N24Q/R35D/R127K/R159K，N24E/G54D/G78D/R159K，R35D/R159K，

R35E/R159K，R127K/R159K，R35E/N67S/G78D/R127Q，

N24E/R35D/G78D，R35D/G78D/R127K/R159E，

N24E/R35E/G54D/N67S/G78D/R127K/R159K，N24T/N67S/R159E，

N24D/R35D/G78D/R159F，N24Q/R35D/N67S/G78D/R127K/R159F，

R35D/G78D/R127Q/R159K，G78D/R159F，N24A/N67S/R159K，

G78D/R127Q/R159K，N24T/G54D/N67S/G78D/R127Y/R159E，

R14I/A63K/G78D/R123F/D184T，R14I/A63K/R123F/R159E/D184T，

R14I/A63K/R123F/R159F/D184T，R14I/A63K/R123F/R159K/D184T，

R14I/A63K/R123F/R159N/D184T，R14I/A63K/R123K/D184T，

R14I/A63K/R123Q/D184T，R14I/A63K/R123Y/D184T，

R14I/A64K/G78D/T86K/T116E/R123F，

R14I/A64K/T86K/T116E/R123F/R159E，

R14I/A64K/T86K/T116E/R123F/R159K，R14I/A64K/T86K/T116E/R123K，

R14I/A64K/T86K/T116E/R123Q，R14I/A64K/T86K/T116E/R123Y，

R14I/G54D/A63K/R123F/D184T，R14I/G54D/A64K/T86K/T116E/R123F，

R14I/G54D/S76N/A93H/R127K/R159K/I181Q，

R14I/G54D/S76V/A93S/R127K/R159K/I181K，

R14I/N24A/A63K/R123F/D184T，R14I/N24A/A64K/T86K/T116E/R123F，

R14I/N24E/A63K/R123F/D184T，R14I/N24E/A64K/T86K/T116E/R123F，

R14I/N24Q/A63K/R123F/D184T，R14I/N24Q/A64K/T86K/T116E/R123F，

R14I/N24T/A63K/R123F/D184T，R14I/N24T/A64K/T86K/T116E/R123F，

R14I/N24TS76N/A93H/R127K/R159K/I181Q，

R14I/N24TS76V/A93S/R127K/R159K/I181K，

R14I/N67AS76N/A93H/R127K/R159K/I181Q，

R14I/N67LS76N/A93H/R127K/R159K/I181Q，

R14I/N67SS76N/A93H/R127K/R159K/I181Q，

R14I/R35D/A64K/T86K/T116E/R123F，

R14I/R35D/S76N/A93H/R127K/R159K/I181Q，

R14I/R35E/A63K/R123F/D184T，R14I/R35E/A64K/T86K/T116E/R123F，

R14I/R35E/S76N/A93H/R127K/R159K/I181Q，

R14I/R35E/S76V/A93S/R127K/R159K/I181K，

R14I/R35K/A63K/R123F/D184T，R14I/S76N/A93H/R127K/R159E/I181Q，

R14I/S76N/A93H/R127K/R159F/I181Q，

R14I/S76N/A93H/R127K/R159N/I181Q，

R14I/S76N/A93H/R127Q/R159K/I181Q，

R14I/S76N/A93H/R127Y/R159K/I181Q，

R14I/S76N/G78D/A93H/R127K/R159K/I181Q，

R14I/S76V/A93S/R127K/R159F/I181K，

R14I/S76V/A93S/R127K/R159N/I181K，

R14I/S76V/A93S/R127Q/R159K/I181K，

R14I/S76V/A93S/R127Y/R159K/I181K，

R14M/G54D/S76N/A93G/R127K/R159K/I181K，

R14M/N24A/S76N/A93G/R127K/R159K/I181K，

R14M/N24E/S76N/A93G/R127K/R159K/I181K，

R14M/N24Q/S76N/A93G/R127K/R159K/I181K，

R14M/N24T/S76N/A93G/R127K/R159K/I181K，

R14M/N67S/S76N/A93G/R127K/R159K/I181K，

R14M/R35D/S76N/A93G/R127K/R159K/I181K，

R14M/R35E/S76N/A93G/R127K/R159K/I181K，

R14M/S76N/A93G/R127K/R159E/I181K，

R14M/S76N/A93G/R127K/R159F/I181K，

R14M/S76N/A93G/R127K/R159N/I181K，

R14M/S76N/A93G/R127Q/R159K/I181K，

R14M/S76N/A93G/R127Y/R159K/I181K and

R14M/S76N/G78D/A93G/R127K/R159K/I181K carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8 and compares the active serine protease variants with raising.In some preferred embodiments, described variant is compared casein hydrolysis (caseinolytic) activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some particularly preferred embodiments, described variant comprises and is selected from following multiple replacement:

R14L/R79T，G12D/R35H/R159E，G12D/R35H/R123Q，

G12D/R35H/R123F/R159E，G12D/R35H，G12D/R35E/R159E，

G12D/R35E/R123Q/R159E，G12D/R35E/R123Q，G12D/R35E，

G12D/R35D/R159E，G12D/R35D/R123Q/R159E，G12D/R35D，

G12D/R159E，G12D/R14Q/R35H，G12D/R14I/R35H，

024E/G049A/A093H/R127K/A143N/R159K/I181Q，

N24M/S76V/A93H/R127K/R159K，

R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T R127A/R159K，

R14I/G65Q and R14I/G65Q/R159K R14I/S76V carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some alternative embodiment, described variant is compared Keratin sulfate hydrolysis (keratinolytic) activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some particularly preferred embodiments, described variant comprises and is selected from following multiple replacement: N024E/G049A/A093H/R127K/A143N/R159K/I181Q,

N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I，

N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N024Q/G049A/A093S/S099N/R127K/A143N/R159K/I181Q，

N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q，

N24A/G54E/S76D/A93G/R127K/R159K，N24E/A93G/R127K/R159K，

N24E/G54L/S76E/A93G/R127K/R159K，N24E/G54Q/A93S/R127K/R159K，

N24E/S76D/A93T/R127K/R159K，N24M/G54E/A93H/R127K/R159K，

N24M/G54E/S76N/A93S/R127K/R159K，N24Q/A93G/R127K/R159K，

N24Q/G54D/S76L/A93G/R127K/R159K，

N24Q/G54I/S76E/A93H/R127K/R159K，

N24T/G54D/S76V/A93G/R127K/R159K，

N24T/G54E/S76V/A93H/R127K/R159K，

N24W/G54D/A93H/R127K/R159K，N24W/S76E/A93G/R127K/R159K，

R014I/S076A/A093G/R127K/R159K/I181T，

R014I/S076A/A093H/R127K/R159K/I181Q，

R014I/S076D/A093H/R127K/R159K/I181Q，

R014I/S076D/A093H/R127K/R159K/I181T，

R014I/S076D/A093S/R127K/R159K/I181T，

R014I/S076E/A093S/R127K/R159K/I181Q，

R014I/S076E/A093T/R127K/R159K/I181K，

R014I/S076I/A093S/R127K/R159K/I181Q，

R014I/S076N/A093H/R127K/R159K/I181Q，

R014I/S076T/A093G/R127K/R159K/I181Q，

R014I/S076V/A093H/R127K/R159K/I181Q，

R014K/S076A/A093S/R127K/R159K/I181T，

R014K/S076E/A093H/R127K/R159K/I181T，

R014K/S076E/A093S/R127K/R159K/I181T，

R014K/S076T/A093H/R127K/R159K/I181Q，

R014L/S076A/A093H/R127K/R159K，

R014L/S076A/A093H/R127K/R159K/I181Q，

R014L/S076D/A093H/R127K/R159K/I181T，

R014L/S076E/A093H/R127K/R159K/I181K，

R014M/S076A/A093G/R127K/R159K/I181T，

R014M/S076A/A093H/R127K/R159K/I181T，

R014M/S076A/A093S/R127K/R159K/I181K，

R014M/S076A/A093S/R127K/R159K/I181T，

R014M/S076A/A093T/R127K/R159K/I181Q，

R014M/S076D/A093S/R127K/R159K/I181T，

R014M/S076E/A093G/R127K/R159K/I181T，

R014M/S076E/A093H/R127K/R159K/I181T，

R014M/S076E/A093S/R127K/R159K/I181T，

R014M/S076N/A093G/R127K/R159K/I181K，

R014M/S076N/A093G/R127K/R159K/I181T，

R014M/S076N/A093H/R127K/R159K/I181T，

R014M/S076N/A093S/R127K/R159K/I181T，

R014M/S076V/A093G/R127K/R159K/I181Q，

R14I/N24Q/A64K/G78D/R123F/R159K/D184T，

R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，

R14I/N24E/A64K/R123F/R127K/R159K/D184T，

R14I/N24Q/A64K/R123F/R127Q/R159K/D184T，

R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T，

R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，R127Q/R159K，

G78D/R127K/R159K，

N67S/G78D/R127K/R159K，R35D/R159K，G78D/R127Q/R159K，

N24A/N67A/R159K，T36S/R127Q/R159E，S15E/T121E/R123Q，

S15E/R35H/R159E，S15E/R35E，S15E/R159E，S15E/R123Q，S15E/R123E，

R79T/R127Q/R179Q，R35H/R159E，R35H/R127Q/R159E，

R35F/R61S/R159Q，R35F/R159Q，R35E/R159E，R35E/R127Q，

R35D/R159E，R35D/R127Q，

R16Q/R79T/R159Q/R179Q，R16Q/R79T/R127Q，R16Q/R79T/R123L，

R16Q/R79T，R16Q/R35F/R123L/R159Q，R16Q/R159Q/R179Q，

R16Q/R127Q/R159Q，R16Q/R123L/R159Q，R14Q/T121E，

R14Q/R35E/T121E，R14Q/R35E/R159E，R14Q/R35E，R14Q/R35D/R127Q，

R14Q/R35D，R14L/R79T/R127Q/R159Q，R14L/R61S/R79T/R123L，

R14L/R35F/R61S，R14L/R127Q/R159Q/R179Q，R14L/R123L/R159Q，

R14I/R35E/R127Q，R14I/R35E/R123E，R14I/R35D/R159E，

R14E/R35H/R127Q，R127Q/R159E，R127Q/R159Q，R123Q/R159E，

R123Q/R127Q/R159E，R123L/R159Q，R123L/R127Q/R159Q，

R123F/R159E，R123E/R127Q/R159E，R123E/R127Q，

G12D/S15E/R35H/R159E，G12D/S15E/R35D，G12D/S15E/R159E，

G12D/S15E，G12D/R35H/T121E/R123Q，

G12D/R35H/R159E，G12D/R35H/R127Q/R159E，G12D/R35H/R123Q，

G12D/R35H/R123F/R159E，G12D/R35H，G12D/R35E/R159E，

G12D/R35E/R123Q/R159E，G12D/R35E/R123Q，G12D/R35E，

G12D/R35D/R159E，G12D/R35D/R127Q，G12D/R35D，G12D/R159E，

G12D/R14Q/R35H，G12D/R14Q/R35D/R123H，G12D/R14Q/R159E，

G12D/R14I/R35H，G12D/R14E，G12D/R127Q/R159E，G12D/R123E/R159E，

R127A/R159K，R14I/G65Q，R14I/G65Q/R127A/R159K，

R14I/G65Q/R159K，R14I/R127A，R14I/R127A/R159K，R14I/R159K，

R14I/R35F，R14I/R35F/G65Q，R14I/R35F/G65Q/R127A/R159K，

R14I/R35F/R159K, R14I/S76V, R14I/T36S/G65Q/R127A/R159K and R35F/R127A/R159K carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8 and compares the active serine protease variants of the scourability with raising.In some preferred embodiments, described variant comprises and is selected from following multiple replacement:

N24F/R159G，/N24F/R159L/R123V，N24I/R127S，/N24L/R159S，

N24S/R159A，N24V/R159L，N24Y/R159F，R14A/N24K/R127S，

R14A/R159W，R14L/N24Y，R14L/R159G，R14L/R159S，R14L/T109M，

R14L/T39P，R14M/R159W，R14S/N24V，R14S/N24Y，R14T/N24A，

R14V/N24G/P189S，R14V/R159W，R127A/R159K，R14I/G65Q，R14I/S76V，

R14I/G65Q/N67L/R159K，R14I/G65Q/R159K，R14I/S76V，R35F/R159Q，

R16Q/R79T，R16Q/R35F，R16Q/R159Q，R14L/R79T，R123L/R159Q，

G12D/S15E，G12D/R35H，G12D/R35D，

N024E/G049A/A093H//R127K/A143N/R159K/I181Q，

N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H，

N024H/S076A/A093T/S099G/R127K/R159K，

N024L/S076V/A093S/S099A/R127K/R159K，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N24E/G54Q/A93S/R127K/R159K，

N24H/A93H/R127K/R159K，N24H/G54L/S76V/A93H/R127K/R159K，

N24L/G54Q/S76A/A93H/R127K/R159K，N24M/A93G/R127K/R159K，

N24M/G54E/A93H/R127K/R159K，N24M/S76V/A93H/R127K/R159K，

N24Q/G54Q/S76T/A93H/R127K/R159K，N24Q/S76A/A93G/R127K/R159K，

N24T/G54Q/S76N/A93G/R127K/R159K，N24T/S76I/R127K/R159K，

N24W/G54D/A93H/R127K/R159K，N24W/S76A/A93H/R127K/R159K，

N24W/S76T/A93G/R127K/R159K，N24W/S76V/A93G/R127K/R159K，

N24W/S76Y/A93G/R127K/R159K，

R014I/S076A/A093G/R127K/R159K/I181T，

R014I/S076A/A093H/R127K/R159K/I181K，

R014I/S076A/A093H/R127K/R159K/I181Q，

R014I/S076A/A093H/R127K/R159K/I181T，

R014I/S076D/A093S/R127K/R159K/I181T，

R014I/S076E/A093T/R127K/R159K/I181K，

R014I/S076N/A093H/R127K/R159K/I181Q，

R014I/S076V/A093H/R127K/R159K/I181Q，

R014I/S076V/A093S/R127K/R159K/I181K，

R014K/S076A/A093S/R127K/R159K/I181T，

R014K/S076E/A093H/R127K/R159K/I181K，

R014K/S076I/A093S/R127K/R159K/I181T，

R014K/S076T/A093H/R127K/R159K/I181K，

R014K/S076T/A093H/R127K/R159K/I181T，

R014K/S076V/A093H/R127K/R159K/I181K，

R014L/S076A/A093H/R127K/R159K，

R014L/S076A/A093H/R127K/R159K/I181Q，

R014L/S076D/A093H/R127K/R159K/I181T，

R014L/S076E/A093H/R127K/R159K/I181K，

R014M/S076A/A093G/R127K/R159K/I181K，

R014M/S076A/A093G/R127K/R159K/I181T，

R014M/S076A/A093H/R127K/R159K/I181T，

R014M/S076A/A093S/R127K/R159K/I181K，

R014M/S076A/A093S/R127K/R159K/I181T，

R014M/S076A/A093T/R127K/R159K/I181Q，

R014M/S076I/A093H/R127K/R159K/I181T，

R014M/S076I/A093S/R127K/R159K/I181T，

R014M/S076N/A093G/R127K/R159K/I181K，

R014M/S076N/A093G/R127K/R159K/I181T，

R014M/S076N/A093H/R127K/R159K/I181T，

R014M/S076N/A093S/R127K/R159K/I181T，

R014M/S076T/A093H/R127K/R159K/I181K，

R014M/S076V/A093G/R127K/R159K/I181Q，

R014M/S076W/A093H/R127K/R159K/I181K，

R014M/S076Y/A093H/R127K/R159K/I181K，

R014M/S076Y/A093H/R127K/R159K/I181T，G012D/R035E/D184N，

G012D/R035E/N067K，R14I/A64K/R123F/R159F/D184T，

R14I/A64K/R123F/R159F/D184T，

R14I/N24Q/A64K/G78D/R123F/R159K/D184T，

R14I/N24Q/A64K/N67S/R123F/R159F/D184T，

R14I/N24Q/A64K/N67A/R123F/R159K/D184T，

R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T，

R14I/A64K/R123F/D184T，R14I/N24A/A64K/R123F/R159N/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，

R14I/N24Q/A64K/N67A/R123F/R159K/D184T，

R14I/N24E/A64K/R123F/R127K/R159K/D184T，

R14I/N24A/A64K/R123F/D184T，R14I/A64K/R123F/R127K/R159F/D184T，

R14I/A64K/R123F/R159N/D184T，

R14I/A64K/R123F/R159K/D184T，

R14I/A64K/R123F/R127Y/R159K/D184T，

R14I/N24Q/A64K/R123F/R127Q/R159K/D184T，

R14I/A64K/R123F/R159K/D184T，

R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T，

R14I/A64K/N67S/G78D/R123F/R127K/R159K/D184T，

R14I/N24Q/A64K/N67A/R123F/R127K/R159K/D184T，R127K/R159K，

N24A/N67S/R159K，N24A/N67A/R159K，R14I/A63K/G78D/R123F/D184T，

R14I/A63K/N67A/R123F/D184T，R14I/A63K/N67L/R123F/D184T，

R14I/A63K/N67S/R123F/D184T，R14I/A63K/R123F/R159F/D184T，

R14I/A63K/R123F/R159K/D184T，R14I/A63K/R123F/R159N/D184T，

R14I/A63K/R123K/D184T，R14I/A63K/R123Q/D184T，

R14I/A63K/R123Y/D184T，R14I/A64K/G78D/T86K/T116E/R123F，

R14I/A64K/N67A/T86K/T116E/R123F，

R14I/A64K/N67L/T86K/T116E/R123F，

R14I/A64K/T86K/T116E/R123F/R159K，R14I/A64K/T86K/T116E/R123K，

R14I/G54D/A63K/R123F/D184T，R14I/G54D/A64K/T86K/T116E/R123F，

R14I/N24A/A63K/R123F/D184T，R14I/N24A/A64K/T86K/T116E/R123F，

R14I/N24A/S76V/A93S/R127K/R159K/I181K，

R14I/N24E/A63K/R123F/D184T，R14I/N24E/A64K/T86K/T116E/R123F，

R14I/N24Q/A63K/R123F/D184T，R14I/N24Q/A64K/T86K/T116E/R123F，

R14I/N24Q/S76V/A93S/R127K/R159K/I181K，

R14I/N24T/A63K/R123F/D184T，R14I/N24T/A64K/T86K/T116E/R123F，

R14I/N24TS76V/A93S/R127K/R159K/I181K，

R14I/N67SS76N/A93H/R127K/R159K/I181Q，

R14I/R35E/A63K/R123F/D184T，R14I/R35K/A63K/R123F/D184T，

R14I/S76V/A93S/R127K/R159F/I181K，

R14I/S76V/A93S/R127K/R159N/I181K，

R14I/S76V/A93S/R127Y/R159K/I181K，

R14M/N24T/S76N/A93G/R127K/R159K/I181K，

R14M/N67S/S76N/A93G/R127K/R159K/I181K，

R14M/S76N/A93G/R127K/R159F/I181K and

R14M/S76N/G78D/A93G/R127K/R159K/I181K carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some embodiments, described variant is compared the performance activity that washes the dishes with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described variant comprises and is selected from following multiple replacement:

R14N/R127K/R159L，R14I/A64K/T86K/N112E/R123F/D184T，

G12D/R35E/G63R/R79K/T109M, R14L, G12D/R35E and

R14M/S76D/A93H/R127K/R159K/I181K carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In other embodiments, described variant is compared the stain with raising and is removed activity with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described variant comprises and is selected from following multiple replacement:

G54E/R14L，N24D/R127Y/R159V，N24E/R127S，N24E/R159C，

N24F/R159G，N24F/R159G/G54E，N24F/R159L/R123V，N24G/R127Y，

N24H/R159Y/T46I，N24I/R127S，N24I/R127V/R14/N24L/R159S，

N24S/R159A，N24V/R127M/R159V，N24V/R127S/R159H，N24V/R159L，

N24Y/G54A，N24Y/R127L，N24Y/R127S，N24Y/R127V，N24Y/R159F，

R127A/R159F，R127H/R159Q，R127H/R159T/S185F，R127M/R159V，

R127S/R159G，R127S/R159L，R127T/R159F，R127V/R159G，R127Y/R159L，

R14A/N24K/R127S，R14A/R127Y/R159W，R14G/N24S/R127C，

R14G/R127G，R14L/N24D，R14L/N24Y，R14L/R123L，R14L/R127S，

R14L/R127V，R14L/R127Y，R14L/R159G，R14L/R159S，R14L/T39P，

R14M/N24L/R159S/R123V，R14M/R159F，R14M/R159W，R14Q/R123F，

R14S/N24E/R127W，R14S/N24V，R14S/N24Y，R14T/N24T/R127Q，

R14T/R127Y，

R14V/N24D/R127C，R14V/N24G/P189S，R14V/N24L/R127F，

R14V/R127A，R14V/R159F，

R14V/R159W，R14W/N24A，R14W/R123L，R14W/R123V，

R14W/R159V，R159V/G49D，

R159V/R123G，N024E/G049A/A093H/R127K/A143N/R159K/I181Q，

N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q，

N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T，

N24A/G54E/S76D/A93G/R127K/R159K，N24E/A93G/R127K/R159K，

N24E/G54L/S76E/A93G/R127K/R159K，N24E/G54Q/A93S/R127K/R159K，

N24E/S76D/A93T/R127K/R159K，N24H/G54E/A93G/R127K/R159K，

N24L/S76T/A93G/R127K/R159K，N24M/A93S/R127K/R159K，

N24M/A93T/R127K/R159K，N24M/G54E/A93H/R127K/R159K，

N24M/G54E/S76N/A93S/R127K/R159K，N24M/S76V/A93H/R127K/R159K，

N24Q/A93G/R127K/R159K，N24Q/G54D/S76L/A93G/R127K/R159K，

N24Q/G54I/S76T/A93G/R127K/R159K，N24Q/G54Q/A93G/R127K/R159K，

N24Q/S76A/A93G/R127K/R159K，N24T/G54Q/S76N/A93G/R127K/R159K，

N24T/G54Q/S76V/R127K/R159K，N24T/S76I/R127K/R159K，

N24W/A93G/R127K/R159K，N24W/G54D/A93H/R127K/R159K，

R014I/S076A/A093G/R127K/R159K/I181T，

R014I/S076A/A093H/R127K/R159K/I181Q，

R014I/S076D/A093H/R127K/R159K/I181Q，

R014I/S076D/A093H/R127K/R159K/I181T，

R014I/S076D/A093S/R127K/R159K/I181T，

R014I/S076E/A093S/R127K/R159K/I181Q，

R014I/S076E/A093T/R127K/R159K/I181K，

R014I/S076N/A093H/R127K/R159K/I181Q，

R014L/S076A/A093H/R127K/R159K，

R014L/S076A/A093H/R127K/R159K/I181Q，

R014L/S076D/A093H/R127K/R159K/I181T，

R014M/S076A/A093G/R127K/R159K/I181T，

R014M/S076A/A093S/R127K/R159K/I181K，

R014M/S076A/A093S/R127K/R159K/I181T，

R014M/S076A/A093T/R127K/R159K/I181Q，

R014M/S076D/A093S/R127K/R159K/I181T，

R014M/S076E/A093H/R127K/R159K/I181T，

R014M/S076E/A093S/R127K/R159K/I181T，

R014M/S076N/A093G/R127K/R159K/I181K，

R014M/S076N/A093G/R127K/R159K/I181T，

R014M/S076N/A093H/R127K/R159K/I181T，

R014M/S076N/A093S/R127K/R159K/I181T，T36S/R127Q/R159E，

S15E/R35E，S15E/R35D，S15E/R159E，S15E/R127Q，S15E/R123Q，

S15E/R123E，R79T/R127Q/R179Q，R35H/R159E，R35F/R61S/R159Q，

R35F/R159Q，R35D/R127Q，R16Q/R79T/R159Q/R179Q，

R16Q/R79T/R123L，R16Q/R79T，R16Q/R35F，R16Q/R159Q/R179Q，

R16Q/R159Q，R14Q/T121E，R14Q/R35E，R14Q/R35D，R14Q/R123Q，

R14L/R79T，R127Q/R159E，R127Q/R159Q，R123Q/R159E，R123L/R159Q，

R123F/R159E，G12D/S15E，G12D/R35H/R159E，G12D/R35H/R123Q，

G12D/R35H，G12D/R35E，G12D/R35D，G12D/R159E，G12D/R14Q/R35H，

G12D/R14I/R35H，G65Q/R127A/R159K，R127A/R159K，R14I/G65Q，

R14I/G65Q/N67L/R159K，R14I/G65Q/R127A，R14I/G65Q/R127A/R159K，

R14I/G65Q/R159K，R14I/R127A，R14I/R127A/R159K，R14I/R159K，

R14I/R35F, R14I/R35F/G65Q, R14I/R35F/R159K, R14I/S76V and R35F/R127A/R159K, wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides the serine protease variants of comparing the surface property with at least a change with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described variant is included in and is selected from following locational at least two replacements: 1,2,4,7,8,10,11,12,13,14,15,16,22,24,25,32,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,57,59,61,62,63,64,65,66,67,68,69,71,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,95,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,123,124,126,127,128,130,131,132,133,134,135,137,143,144,145,146,147,148,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,173,174,175,176,177,178,179,180,181,182,183 and 184, wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.

In some particularly preferred embodiments, described misfolded proteins enzyme is compared with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8 has at least a improved characteristic.In some particularly preferred embodiments, described at least a improved characteristic is selected from acid acceptance, thermostability, casein hydrolysis, Keratin sulfate hydrolysis, clean-up performance and LAS stability.

The present invention also provides the expression vector of the polynucleotide sequence that comprises the encoding serine ease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.The present invention also provides the host cell that comprises at least a expression vector, in some preferred embodiments, described host cell is genus bacillus (Bacillus.sp), and in other other embodiments, described host cell is streptomycete (Streptomyces.sp), and in other embodiments, described host cell is aspergillus (Aspergillus.sp), in other embodiments, described host cell is a wood mould (Trichoderma.sp).The serine protease variants that the present invention also provides described host cell to produce.

The present invention also provides the composition that comprises the serine protease variants that at least a portion this paper provided, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and wherein said replacement is carried out on the position of the Cellulomonas 69B4 proteolytic enzyme position that is equivalent to comprise aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides the polynucleotide sequence of encoding serine ease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.The present invention also provides the expression vector of the polynucleotide sequence that comprises the encoding serine ease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.The present invention also provides the host cell that comprises at least a expression vector, in some preferred embodiments, described host cell is a genus bacillus, and in other embodiments, described host cell is a streptomycete, and in other embodiments, described host cell is an aspergillus, in other embodiments, described host cell is that wood is mould.The serine protease variants that the present invention also provides described host cell to produce.

The present invention also provides the cleaning compositions that comprises serine protease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described cleaning compositions comprises at least a variant serine protease, and wherein said serine protease has the immunology cross reactivity to serine protease shown in the SEQ ID NO:8.In some preferred embodiments, described replacement is in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8 1,2,3,4,7,8,9,10,11,12,13,14,15,16,18,19,22,24,25,26,27,28,29,30,31,32,33,34,35,36,38,39,40,41,42,43,44,45,46,47,48,49,51,52,54,55,56,57,59,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,96,99,100,101,103,104,105,107,109,110,111,112,113,114,115,116,117,118,119,121,123,124,125,126,127,128,129,130,132,133,134,135,136,137,140,141,142,143,144,145,146,147,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,172,175,176,177,179,180,181, carry out on 182,183,184,185,186,187,188 and 189 bit positions.In some preferred embodiments, described cleaning compositions also comprises one or more other enzymes or enzyme derivatives, and it is selected from proteolytic enzyme, amylase, lipase, mannase (mannanase), polygalacturonase, at (cutinases), oxydo-reductase, hemicellulase and cellulase.

The present invention also provides the composition that comprises serine protease variants and at least a stablizer, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described composition is a cleaning compositions.In some particularly preferred embodiments, described stablizer is selected from borax, glycerine and competitive inhibitor.In other particularly preferred embodiments, described competitive inhibitor makes this serine protease variants stable to aniorfic surfactant.In other embodiments, described serine protease variants is the stable variant of self-dissolving.

The present invention also provides serine protease variants and the optional cleaning compositions that comprises supplementary component that comprises at least 0.0001 weight percent, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.In some preferred embodiments, described composition comprises the about 0.001 at least a serine protease variants to about 0.5 weight percent.In some preferred embodiments, described composition comprises about 0.01 serine protease to about 0.1 weight percent.In other embodiment preferred, described cleaning compositions comprises supplementary component.In other embodiment preferred, described cleaning compositions comprises the pH regulator agent of capacity, thinks that said composition provides about 3 to about 5 clean pH, and wherein said composition does not contain the material of hydrolysis under about 3 to about 5 pH substantially.In some alternative embodiment preferred, described cleaning compositions comprises the material of hydrolysis, comprises surfactant material.In some particularly preferred embodiments, described surfactant material comprises the alkylsurfuric acid natrium surfactant that contains ethylene oxide moiety.

The present invention also provides the cleaning compositions that comprises serine protease variants, described serine protease variants has the aminoacid sequence that comprises at least two amino acid replacements, carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8, wherein said cleaning compositions is a liquid.In some alternative embodiment, described cleaning compositions is powder, particle or tablet composition.

The present invention also provides the cleaning compositions that comprises serine protease variants, described serine protease variants has the aminoacid sequence that comprises at least two amino acid replacements, carry out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8, wherein said cleaning compositions also comprises hydrogen peroxide cource.In some preferred embodiments, described catalase source comprises at least a persalt, and wherein said persalt is alkali metal perborate, alkali metal percarbonate, basic metal superphosphate, alkali metal persulphate or its mixture.In some particularly preferred embodiments, described cleaning compositions also comprises bleaching catalyst, bleach activator and/or its mixture.

The present invention also provides cleaning method, and it may further comprise the steps: the surface and/or the article that a) will comprise fabric contact at least a cleaning compositions of the present invention; And b) randomly the washing and/or described surface of rinsing or material.

The present invention also provides the animal-feed that comprises serine protease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides fabric and/or the leather processing composition that comprises serine protease variants, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

The present invention also provides the Personal hygiene that comprises serine protease variants composition, described serine protease variants has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

The accompanying drawing summary

Fig. 1 provides the figure of plasmid pHPLT-ASP-C1-2.

Fig. 2 provides the figure of plasmid pXX-KpnI.

Fig. 3 provides the figure of plasmid pHPLT.

Fig. 4 provides the figure of plasmid pUC18.

Fig. 5 provides the figure of plasmid pUC18-ASP.

Fig. 6 provides the form that is presented at the tergotometer test result of carrying out under the situation that does not have the HEPES damping fluid (the enzyme amount is 0.55ppm).

Fig. 7 provides the form that is presented at the tergotometer test result of carrying out under the situation that has the HEPES damping fluid (the enzyme amount is 0.55ppm).

Detailed Description Of The Invention

The invention provides the new micrococcus luteus serine protease that contains multiple replacement. Particularly, the invention provides the serine protease that contains multiple replacement, these protease of encoding DNA, comprise the DNA of this protease of encoding carrier, transform the enzyme that host cell that described carrier DNA is arranged and this host cell produce. The present invention also provides cleaning composition (as the washing agent composition), animal feed composition and fabric and the leather processing compositions that comprises these serine protease variants. In particularly preferred embodiments, the invention provides mutant (being variant) protease from wild-type protease described herein. These misfolded proteins enzymes also can be used for multiple application.

The invention provides the misfolded proteins enzyme with multiple replacement. Importantly, these variant enzymes have good stability and proteolytic activity. These variant enzymes can be used for multiple application, include but are not limited to cleaning composition, animal feed, fabric processing etc. The present invention also provides the method that produces these enzymes. In some preferred embodiments, misfolded proteins enzyme of the present invention is pure or relatively pure form.

The present invention also provides the nucleotide sequence that is suitable for producing misfolded proteins enzyme of the present invention in the restructuring biology. In some embodiments, restructuring produces provides with the change method of body protein enzyme of commercial feasible volume production.

Except as otherwise noted, enforcement of the present invention relates to routine techniques commonly used in molecular biology, microbiology and recombinant DNA, and they are within the technology of this area. These technology are that those skilled in the art are known, and be described in multiple textbook and list of references (is consulted as Sambrook etc., " Molecular Cloning:A Laboratory Manual ", second edition (Cold Spring Harbor), [1989]); With Ausubel etc., " Current Protocols in Molecular Biology " [1987]). All patents, patent application, article and publication that this paper mentions are all clearly incorporated this paper into as a reference.

Unless indicate in addition herein, all scientific and technical terminologies used herein all have those skilled in the art's the identical implication of common understanding. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons, NY (1994) and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide the general dictionary of many terms used herein for those skilled in the art. , although any method and material similar with material to methods described herein or that be equal to all can be used for implementing the present invention, this paper describes preferred method and material. Therefore, hereinafter the term of definition will be able to more complete description with reference to specification integral body. Simultaneously, odd number " " and " this " include plural implication, unless its context clearly represents not to be like this. Number range comprises the numerical value that defines this scope. Except as otherwise noted, otherwise nucleic acid writes from left to right with 5 ' to 3 ' direction, and amino acid sequence is write from left to right with amino to the direction of carboxyl. Should be appreciated that, the present invention is not subject to described concrete grammar, scheme and reagent, and they can use their situation to change according to those skilled in the art.

Except as otherwise noted, otherwise the routine techniques of protein purification, molecular biology, microbiology, recombinant DNA technology and protein sequencing is used in enforcement of the present invention, and they are all within the technology of this area.

In addition, title provided herein does not limit many aspects of the present invention or embodiment, and they can be whole with reference to specification. Therefore, hereinafter the term of definition can more completely be defined with reference to specification integral body. However,, for the ease of understanding the present invention, numerous terms have hereinafter been defined. Other are defined in U.S. Patent application sequence number 10/576,331 incorporated herein by reference and provide.

I. definition

Term used herein " protease " and " proteolytic activity " refer to protein or the peptide of the ability of the substrate that shows the hydrolysis peptide or have peptide bond. The multiple method of knowing (Kalisz, " Microbial Proteinases for measuring proteolytic activity is arranged, " Fiechter (editor),Advances in Biochemical Engineering/Biotechnology, [1988]). The comparative measurements of ability that for example, can be by analyzing each protease hydrolytic commodity substrate confirms proteolytic activity. The exemplary substrate that can be used for analyzing proteins enzyme or proteolytic activity includes but are not limited to dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), ox elastin laminin (Sigma E-1625) and ox horn albumen (ICN Biomedical 902111). Using the colorimetric estimation that these substrates carry out is (consult as WO 99/34011 and U.S. Patent number 6,376,450, all incorporate this paper into as a reference) known in the art. PNA measures (consult as Del Mar etc., Anal.Biochem., 99:316-320[1979]) and also can be used for the fraction of collecting in the gradient elution process is measured organized enzyme concentration. The speed of liberate p-nitroaniline when these determination methods are measured the synthetic substrate succinyl-Ala-Ala of enzyme hydrolysis solubilities-proline-phenylalanine-to p-nitroanilide (sAAPF-pNA). Measuring in hydrolysis and producing yellow speed at the 410nm place on spectrophotometer, itself and organized enzyme concentration are proportional. In addition, can measure total protein concentration with the absorbance measuring at 280nm place. The ratio of organized enzyme/total protein has provided the purity of enzyme.

Term used herein " ASP protease ", " Asp protease " and " Asp " refer to serine protease described herein. In some preferred embodiments, described Asp protease is the protease that derives from fiber aeromonas strain 69B4 of called after 69B4 protease in this article. Therefore, in preferred embodiments, term " 69B4 protease " refers to the naturally occurring maturation protein enzyme from fiber aeromonas strain 69B4 (DSM 16035), and it has the essentially identical amino acid sequence with SEQ ID NO:8. In alternative embodiment, the invention provides the part of ASP protease.

Term " fiber monad protease homology thing " refers to have the native protein enzyme with the essentially identical amino acid sequence of maturation protein enzyme from fiber aeromonas strain 69B4, perhaps the encode polynucleotide sequence of these natural protease, described protease keeps the functional character of these nucleic acid institute encoding serine protease. In some embodiments, these protease homology things are called " fiber monad protease (cellulomonadins) ". Term used herein " ease variants ", " ASP variant ", " ASP ease variants " and " 69B ease variants " are used in reference to such protease of generation, it is similar to wild type ASP, particularly similar in function aspects, but contain sudden change (for example replacing) in its amino acid sequence, make it be different from wild-type protease on sequence. Some identify that the amino acid residue that is used for replacing is the conservative residue, and other are not. In some embodiments, ease variants of the present invention comprises the mature form of ease variants, and in other embodiments, the invention provides precursor form and the front former form of these ease variants.

" fiber monad " used herein refers to all kinds in " Cellulomonas (Cellulomonas) ", it is gram-positive bacteria, be included into actinomyces (Actinobacteria) guiding principle, Actinomycetal (Actinomycetales), micrococcus luteus (Micrococcineae) suborder, fiber unit cell Cordycepps (Cellulomonadaceae). People are generally acknowledged, and Cellulomonas is still in experience classification restructuring. Therefore, this genus is intended to comprise the species that reclassified.

" streptomycete (Streptomyces ssp.) " used herein refers to all species in " streptomyces (Streptomyces) ", it is gram-positive bacteria, be classified as Actinomycetes, Actinomycetal, streptomycete (Streptomycineae) suborder, Streptomycetaceae (Streptomycetaceae). People are generally acknowledged, and streptomyces is still in experience classification restructuring. Therefore, this genus is intended to comprise the species that reclassified.

term used herein " bacillus (Bacillus) genus " refers to any those skilled in the art's known species in " bacillus ", include but are not limited to bacillus subtilis (B.subtilis), bacillus licheniformis (B.licheniformis), bacillus lentus (B.lentus), bacillus brevis (B.brevis), bacillus stearothermophilus (B.stearothermophilus), basophilic bacillus (B.alkalophilus), bacillus amyloliquefaciens (B.amyloliquefaciens), Bacillus clausii (B.clausii), salt tolerant bacillus (B.halodurans), bacillus megaterium (B. megaterium), bacillus coagulans (B.coagulans), Bacillus circulans (B.circulans), bacillus lautus (B.lautus) and bacillus thuringiensis (B.thuringiensis). people are generally acknowledged, and bacillus is still in experience classification restructuring. therefore, this genus is intended to comprise the species that reclassified, and includes but are not limited to such as bacillus stearothermophilus (now being called " thermophilic fatty ground bacillus (Geobacillus stearothermophilus) "). produce the resistance endospore and be considered to the restriction feature of bacillus under oxygen exists, although this feature also is applicable to the ring grease acid bacillus (Alicyclobacillus) of name recently, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, happiness salt bacillus (Halobacillus), series bacillus belongs to (Paenibacillus), Salibacillus, Thermobacillus, separate urea bacillus (Ureibacillus) and twig spore Bacillus (Virgibacillus).

Term " polynucleotides " and " nucleic acid " are used interchangeably in this article, refer to the polymerized form of the nucleotides (ribonucleotide or deoxyribonucleotide) of any length. These terms include but not limited to DNA, genomic DNA, cDNA, RNA, the DNA RNA hybrid of strand or double-stranded or three chains, perhaps comprise purine and pyrimidine bases or other polymers natural, that modify through chemistry or biochemistry, non-natural or derivative nucleotide base. Be below the limiting examples of polynucleotides: RNA, nucleic acid probe and the primer of the DNA of any sequence of gene, genetic fragment, chromosome segment, EST, extron, introne, mRNA, tRNA, rRNA, ribozyme, cDNA, restructuring polynucleotides, side chain polynucleotides, plasmid, carrier, separation, any sequence of separation. In some embodiments, polynucleotides comprise modified nucleotides (for example methylated nucleotide) and nucleotide analog, uracil, other sugar and linking group such as fluoro ribose and thioester (thioate) and nucleotides branch. In alternative embodiment, nucleotide sequence is interrupted by the non-nucleotide component.

Term used herein " gene " refers to polynucleotides (as the DNA section), its coded polypeptide, and the intervening sequence (introne) between before comprising code area and zone afterwards and each coding section (extron).

" homologous gene " used herein refers to that they correspond to each other, and mutually the same or highly similar from the one pair of genes of species different but that usually be correlated with. This term comprises the gene (for example, orthologous gene) that is separated by species formation (being the generation of new species) and the gene (as the collateral line homologous gene) that is separated by gene duplication.

" homology " used herein refers to similitude or the homogeneity of sequence, preferably refers to homogeneity. This homology use standard technique known in the art determine (consult as Smith and Waterman, Adv.Appl.Math., 2:482[1981]; Needleman and Wunsch, J.Mol.Biol., 48:443[1970]; Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444 [1988]; GAP, BESTFIT, FASTA and TFASTA in program such as Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); And Devereux etc., Nucl.Acid Res., 12:387-395[1984]).

" similar sequence " used herein is such sequence, and wherein the function of this gene is with basic identical based on the gene of fiber aeromonas strain 69B4 protease. In addition, similar gene and fiber aeromonas strain 69B4 protease comprise at least 45%, 50%, and 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence homogeneity. Perhaps, gene 70 to 100% couplings in similar sequence and fiber aeromonas strain 69B4 protease district, and/or contain 5-10 gene at least in the zone with fiber aeromonas strain 69B4 chromogene coupling. In other embodiments, more than one in above characteristic are applicable to this sequence. Similar sequence is determined by known sequence alignment method. Comparison method commonly used is BLAST, but as above hereinafter point out, additive method also can be used for aligned sequences.

" restructuring " used herein comprises phalangeal cell or carrier, its by introduce the allos nucleotide sequence and carried out modifying or this cell derived in the cell of such modification. Therefore, for example, due to the human intervention of having a mind to, recombinant cell is expressed the gene that with identical form, does not exist in natural (non-restructuring) form cell, perhaps expresses original abnormal expression, the low natural gene of expressing or not expressing fully. " restructuring " and generation " restructuring " nucleic acid are normally assembled two or more nucleic acid fragments, and wherein this assembling produces mosaic gene.

In preferred embodiments, be created at least one codon place and carry out the sudden change DNA sequence dna of site saturation mutagenesis. In another preferred embodiment, two or more codons are carried out the site saturation mutagenesis. In another embodiment, sudden change DNA sequence dna and wild-type sequence have higher than 50%, higher than 55%, higher than 60%, higher than 65%, higher than 70%, higher than 75%, higher than 80%, higher than 85%, higher than 90%, higher than 95% or higher than 98% homology. In alternative embodiment, sudden change DNA uses any known mutagenesis method to produce in vivo, such as radiation, nitrosoguanidine etc. Then separate the DNA sequence dna of expectation and be used for method provided herein.

" amino acid " used herein refers to peptide or protein sequence or its part.

" purpose albumen " used herein and " purpose polypeptide " refer to the protein/polypeptide of expecting and/or assessing. In some embodiments, purpose albumen is at intracellular expression, and in other embodiments, purpose albumen is the polypeptide of secretion. In particularly preferred embodiments, these enzymes comprise serine protease of the present invention. In some embodiments, purpose albumen is the secrete polypeptide that merges with signal peptide (aminoterminal of namely treating secretory protein extends). Nearly all secretory protein all utilizes the protein of aminoterminal end to extend, and brings into play key effect in its target in the precursor protein cross-film and transhipment. This extending in the film transport process or after transhipment excised by proteolysis by signal peptidase immediately.

Term used herein " allos albumen " refers to not naturally occurring protein or polypeptide in host cell. The example of allos albumen comprises enzyme,, as hydrolase, comprises protease. In some embodiments, the gene of this albumen of encoding is naturally occurring gene, and in other embodiments, uses sudden change and/or synthetic gene.

Natural or naturally occurring protein or polypeptide in " homologous protein " used herein phalangeal cell. In preferred embodiments, described cell is gram-positive cell, and in particularly preferred embodiments, this cell is the bacillus host cell. In alternative embodiment, homologous protein is the native protein that other biological (including but are not limited to Escherichia coli, streptomycete, the mould and aspergillus of wood) produces. The present invention includes the host cell that produces homologous protein by recombinant DNA technology.

Term " protein " and " polypeptide " are used interchangeably in this article. In present disclosure, a full piece of writing all uses 3 letter amino acid codes consistent with IUPAC-IUB biochemical nomenclature commission (JCBN). Should be appreciated that, polypeptide can be due to the degeneracy of genetic code by more than a kind of nucleotide sequence coded.

" naturally occurring enzyme " refers to have the enzyme of the unmodified amino acid sequence identical with occurring in nature. Naturally occurring enzyme comprises natural enzyme, by specific microorganism, is expressed or be present in wherein those enzymes.

Term " from " and " deriving from " protease of not only referring to by or can being produced by the biological bacterial strain of purpose, also refer to by the DNA sequence encoding that separates from these bacterial strains and the protease that produces in containing the host living beings of this type of DNA sequence dna. In addition, this term refers to by the DNA sequence encoding in synthetic and/or cDNA source and has the protease of the described identification mark of purpose protease. For example, " from the protease of fiber monad " refers to have the enzyme of the proteolytic activity of the natural generation of fiber monad, and such serine protease, it is similar to the enzyme that fiber monad source produces, but uses genetic engineering technology that biological generation of non-fiber monad of the nucleic acid of this serine protease of coding arranged by conversion.

" derivative " in this range of definition keeps the characteristic proteolytic activity of observing in wild type, natural or parent's form usually, and its reserving degree makes this derivative can be used for and described wild type, natural or parent's form similar purpose. That the functional derivatives of serine protease comprises is naturally occurring, synthetic or restructuring produces peptide or fragments of peptides, it has the general features of serine protease of the present invention.

Term " functional derivatives " refers to the derivative of nucleic acid, and it has the functional characteristic of the nucleic acid of encoding serine protease. That the functional derivatives of the nucleic acid of code book invention serine protease comprises is naturally occurring, synthetic or restructuring produces nucleic acid or fragment, and the characteristic serine protease of the present invention of encoding. The wild-type nucleic acid of code book invention serine protease comprises naturally occurring allele and based on the homologue of genetic code degeneracy known in the art.

Term " identical " is in the situation that two nucleic acid or peptide sequence refer to take the highest correspondence as purpose after comparison residue identical in two sequences, this with following sequence relatively or one of parser measure.

Term " best comparison " refers to that this comparison provides the highest percentage homogeneity mark.

With regard to two amino acid, polynucleotides and/or gene orders (suitably), " percentage sequence homogeneity ", " percentage amino acid sequence identity ", " percentage gene order homogeneity " and/or " percentage nucleic acid/polynucleotide sequence homogeneity " refer to the percentage of described sequence identical residue in rear two sequences of the best comparison. Therefore, to be illustrated in the peptide sequences of two best comparisons 80% amino acid be identical to 80% amino acid sequence identity.

Like this, phrase " basic identical " is in the situation that two nucleic acid or polypeptide refer to that use program or algorithm are (as BLAST, ALIGN, CLUSTAL) comprise at least 70% sequence homogeneity, preferred at least 75%, preferred at least 80%, preferred at least 85%, preferred at least 90%, preferred at least 95%, preferred at least 97%, preferred at least 98% and preferably polynucleotides or the polypeptide of at least 99% sequence homogeneity while using canonical parameter with the reference sequences comparison. The essentially identical index of two polypeptide is that first polypeptide and second polypeptide have the immunology cross reactivity. Usually, different polypeptide has the immunology cross reactivity due to the conservative amino acid replacement. Therefore, only not simultaneously, a polypeptide and another polypeptide are basic identical due to the conservative replacement when two polypeptide. Two essentially identical another indexs of nucleic acid sequence are (for example in the paramount stringent condition under) hybridization each other under stringent condition of these two molecules.

Term " be equal to " refer in this article by in the serine protease that can encode with the polynucleotides with multi-nucleotide hybrid of sequence shown in SEQ ID NO:1 under paramount stringent condition. For example, the ripe serine protease that the expression that is equal to is equal to comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and/or at least 99% sequence homogeneity with the mature fibers monad serine protease with amino acid sequence shown in SEQ ID NO:8.

Term " separation " or " purifying " refer to from the material of its primal environment (for example,, if naturally occurring, being its natural surroundings) from shifting out. For example, if material exists with the concentration higher than in natural or wild-type biology in specific composition, perhaps with express in natural or wild-type biology after common non-existent component combination, it be " purifying ". For example, the natural polynucleotides or the polypeptide that are present in live animal do not separate, but identical polynucleotides or polypeptide with this natural system in the some or all of materials that coexist separate after separating. Such polynucleotides can be the parts of carrier, and/or such polynucleotides or polypeptide can be the part of composition, but still separate, because examples of such carriers or composition are not the parts of its natural surroundings. For example, in preferred embodiments,, if nucleic acid or protein provide a band substantially in electrophoresis or trace, claim that it is purifying.

When relating to the DNA sequence dna use, term " separation " refers to such DNA sequence dna, it shifts out from its natural genotypic environment, therefore do not contain other external or undesired coded sequences, and for be suitable for the form of using in the protein generation system through the heredity transformation. The molecule of such separation is the molecule that separates from its natural surroundings, comprises cDNA and genomic clone. The DNA molecular of separation of the present invention does not contain other genes that usually are associated with it, but may comprise naturally occurring 5 ' and 3 ' non-translational region, for example promoter and terminator. The evaluation of relevant range is (consulting as Dynan and Tijan Nature 316:774-78 [1985]) clearly for those of ordinary skills. Term " DNA sequence dna of separation " is also referred to as " clone's DNA sequence dna ".

When relating to the protein use, term " separation " refers to the protein that exists under the condition beyond its natural surroundings. In a kind of preferred form, the protein of separation does not contain other protein, particularly other homologous proteins substantially. While by SDS-PAGE, measuring, the purity of the protein of separation is higher than 10%, preferably higher than 20%, even more preferably higher than 30%. Other aspects of the present invention comprise the protein that is determined as High Purity form (be purity higher than 40%, higher than 60%, higher than 80%, higher than 90%, higher than 95%, higher than 97%, even higher than 99%) by SDS-PAGE.

Term used herein " combinatorial mutagenesis " refers to produce the method in the variant library of homing sequence. In these libraries, variant contains the one or more sudden changes (for example replacing) that are selected from predetermined sudden change group. In addition, the method provides the not method of the random mutation in predetermined sudden change (as replacing) group of introducing. In some embodiments, the method comprises disclosed method in the U.S. Patent application sequence number 09/699.250 of submitting on October 26th, 2000, and this application is incorporated this paper into as a reference. In alternative embodiment, the combinatorial mutagenesis method (for example comprises commercially available kit

Multisite， Stratagene，San Diego，CA)。

Term used herein " mutant library " refers to that genome is most of identical but comprise the cell mass of the different homologues of one or more genes. Such library can be used for for example identifying to have gene or the operon that improves proterties.

Term used herein " initial gene " refers to encode, and the present invention to be used improves and/or the purpose gene of the purpose albumen that changes.

Term used herein " multiple sequence comparison (multiple sequence alignment, MSA) " refers to use the sequence of multiple homologue of the initial gene of algorithm (as Clustal W) comparison.

Term used herein " consensus sequence " and " example sequence " refer to the prototype sequence that all variants of specified protein or purpose sequence are compared with it. This term also refers to show the sequence of the nucleotides that the most often exists in the purpose DNA sequence dna. For each position of gene, consensus sequence is given in MSA the highest amino acid of abundance on this position.

Term used herein " total sudden change " refers to the sequence difference between initial gene and consensus sequence. Have sudden change by the sequence with initial gene and from MSA gained consensus sequence, compare to identify. In some embodiments, will have sudden change introduces in initial gene, so that it is more similar to consensus sequence. Total sudden change also comprises such amino acid change: this change with the amino acid change in initial gene become find in MSA this position upper frequency higher than initial gene in the amino acid of amino acid whose frequency. Therefore, term " total sudden change " comprises with the amino acid substitution in initial gene being that amino acid whose all single amino acids abundanter than amino acid in MSA change.

Term used herein " initially hits " and refers to make up by screening the variant that identify in total mutagenesis library. In preferred embodiments, initially hit to compare with initial gene and have improved performance characteristic.

Term used herein " improvement is hit " refers to the variant that identify in the total mutagenesis library of the combination that strengthens by screening.

Term used herein " improves sudden change " and " performance enhancing sudden change " (" improve and replace " and " performance strengthens replacement ") refers to the sudden change that causes performance to improve when introducing initial gene (as replacing). In some preferred embodiments, these sudden changes (as replacing) check order to identify by hitting of identifying in the screening step to the method. In most embodiments, the sudden change that more frequently occurs in hitting (as replacing) is compared with the total mutagenesis library of the combination of not screening and is likely improvement sudden change (as replacing).

Term used herein " combination of enhancing has the mutagenesis library " refers to based on the screening of the CCM mutagenesis of round before and screening and/or sequencing result and the CCM library of designing and building. In some embodiments, the sequence that before the CCM library of enhancing is based on, the CCM gained of round initially hits. In other embodiments, the CCM that strengthens of design is to be conducive to more common sudden change in initially hitting in the mutagenesis of round before and screening. In some preferred embodiments, this primer that reduces sudden change by omitting coding efficiency is realized, perhaps by the concentration of using other primers to improve the primer of coding efficiency enhancing sudden change in the CCM library with respect to before, realizes.

Term used herein " performance reduces sudden change " refers to the sudden change in the total mutagenesis library of combination, and its frequency in the screening gained hits is compared lower with the total mutagenesis library of the combination of not screening. In preferred embodiments, the abundance of the variant that contains " performance reduces sudden change " is removed and/or reduced to screening technique.

Term used herein " functional assays " refers to provide the mensuration of the index of protein active. In particularly preferred embodiments, this term refers to the mensuration system of analysing protein with its common ability performance function. For example, for the situation of enzyme, functional assays comprises measures the validity of this enzyme aspect catalytic reaction.

Term used herein " target characteristic " refers to the characteristic of initial gene to be changed. The present invention is not intended to only limit to any specific target characteristic. Yet in some preferred embodiments, the target characteristic is the stability resistance of sex change, proteolysis or other degradation factors (for example to) of gene outcome, and in other embodiments, has changed the level of production in producing the host. In fact, any characteristic of initial gene all can be used for the present invention.

Term used herein " characteristic " or its grammer equivalent word are in the situation that nucleic acid refers to any characteristic or the attribute that can select or detect of nucleic acid. These characteristics include but are not limited to affects characteristic, the characteristic of giving the cell that comprises specific nucleic acid of with polypeptide, being combined, the characteristic (as promoter intensity, promoter identification, promoter adjusting, enhancer function) that affects genetic transcription, the characteristic (as RNA montage, rna stability, RNA conformation and posttranscriptional modification) that affects RNA processing, affect the characteristic (for example combination of level, adjusting, mRNA and ribosomal protein, posttranslational modification) of translating. For example, can change the binding site of the transcription factor, polymerase, regulatory factor etc. of nucleic acid, with the feature that produces expectation or identify the feature of not expecting.

Term used herein " characteristic " or its grammer equivalent word are in the situation that polypeptide refers to any feature of selecting or detecting or the attribute of polypeptide. These characteristics include but are not limited to oxidation stability, substrate specificity, catalytic activity, heat endurance, alkaline stability, pH activity profile, the resistance to proteolytic degradation, K_M、k _cat、k _cat/k _MThe ability of ratio, protein folding, induce immune response, binding partner, ability, the secretion capacity of bind receptor, the ability that is showed in cell surface, oligomerization ability, signal ability, the ability that stimulates cellular proliferation, suppress cell proliferation ability, induce the ability of apoptosis, the ability of modifying by phosphorylation or glycosylation, the ability for the treatment of disease.

Term used herein " screening " has the common implication of this area, is generally the multi-step process. In the first step, provide mutant nucleic acid or its variant polypeptide. In second step, measure the characteristic of this mutant nucleic acid or variant polypeptide. In the 3rd step, with surveys characteristic and accordingly the characteristic of characteristic, the corresponding natural polypeptides of precursor nucleic acid or the characteristic that produces the parent material (as initial sequence) of this mutant nucleic acid compare.

It is apparent that to those skilled in the art, the screening technique that be used for to obtain to have the nucleic acid that changes characteristic or protein depends on the characteristic of parent material will promote its modification by producing this mutant nucleic acid. Therefore, skilled person in the art will appreciate that the present invention is not subject to any concrete property to be screened, following characteristic description has only been listed illustrative example. For the method for screening any specific characteristic, general the description arranged in the art. For example, can be in sudden change fore-and-aft survey combination, pH, specificity etc., variation wherein shows and has produced change. Preferably, in the high flux mode, screen, comprise and screen simultaneously a plurality of samples, include but are not limited to the mensuration of using chip, phage display and multiple substrate and/or indicator to carry out.

In some embodiments, screening used herein comprises the selection step, wherein enrichment purpose variant from the variant group. The example of these embodiments comprises the variant of selecting to give the host living beings growth vigor, and phage display or any other displaying method, wherein variant can based on its in conjunction with or catalysis characteristics and being hunted down from the variant group. In preferred embodiments, the variant library is exposed to coerce (such as heat, protease, sex change etc.), identify in screening thereafter still complete variant or by selection come enrichment they. This term is intended to comprise any suitable system of selection. In fact, the present invention is not intended to be limited to any specific screening technique.

Term used herein " target randomization " refers to produce the method for the multiple sequence that wherein one or more positions have been randomized. In some embodiments, randomization is completely random (that is, four kinds of all base A, T, G and C can appear at the randomization position). In alternative embodiment, the randomization of nucleotides only limits to a subgroup of these four kinds of nucleotides. The target randomization can be applicable to one or more codons of sequence, one or more purpose albumen of encoding. During expression, the library of gained produces the protein group, and wherein one or more amino acid positions can contain the mixture of all 20 seed amino acids or amino acid subgroup, and this randoming scheme by the randomization codon determines. In some embodiments, the individual member in target randomization gained colony is due to the target of codon or insert at random or there is amino acid no purpose difference in disappearance. In other embodiments, the protein group who produces comprises synthetic amino acid. In some preferred embodiments, the most members in target randomization gained colony compare with initial gene with consensus sequence and show higher sequence homology. In some embodiments, one or more purpose albumen of described sequential coding. In alternative embodiment, described protein has different biological functions. In some preferred embodiments, enter sequence and comprise at least one selected marker.

Term " modification sequence " and " modifier " are used interchangeably in this article, refer to comprise the sequence of disappearance, insertion or the interruption of natural acid sequence. In some preferred embodiments, the expression product of modification sequence is the protein (if modify, being for example, disappearance or the interruption of sequence) of brachymemma. In some particularly preferred embodiments, the protein retains biological activity of described brachymemma. In alternative embodiment, the expression product of modifying sequence is the protein (for example, comprising the modification of the insertion in nucleotide sequence) that extends. In some embodiments, insert the protein cause brachymemma (for example, when insertion cause forming the termination codon period of the day from 11 p.m. to 1 a.m). Therefore, insertion can produce the albumen of truncated protein or prolongation as expression product.

Term used herein " replacement " comprises another amino acid for this position in this amino acid sequence of an amino acid substitution in amino acid sequence. Therefore, this term comprises that another amino acid whose insertion in amino acid sequence and/or disappearance have changed the situation of the relative position of this amino acid in this sequence. Therefore, although amino acid may be by the specificity target, be used for replacing (for example using methods described herein), this amino acid may be due to the variation of its relative position in this sequence another amino acid " replacement " in by sequence. For example, by inserting amino acid between 1 and 2 bit amino acid in SEQ ID NO:8, the sequence of gained will move an amino acid (that is, originally the amino acid on 2 is positioned at 3 etc. now). This term is intended to comprise a plurality of and single change (being single replacement or multiple replacement) in amino acid sequence. It shall yet further be noted that amino acid substitution used herein represents like this: natural amino acid is thereafter position, thereafter the amino acid for replacing. Therefore, in N024E (also being designated as N24E) expression SEQ ID NO:8, the asparagine of 24 is replaced by glutamic acid. As described herein, multiple replacement represents with "/" or "-". Therefore, " R127A-G65Q " and " R127A/G65Q " represents that all 127 and 65 s' amino acid is replaced (that is, the arginine of 127 is replaced by alanine, and the glycine of 65 is replaced by glutamine).

Term used herein " mutant nucleotide sequence " and " mutator " are used interchangeably, and refer to contain at least one codon place of host cell wild-type sequence the sequence that changes. The expression product of mutant nucleotide sequence is to contain the protein of the amino acid sequence that changes with respect to wild type. Expression product can have the Functional Capability (enzymatic activity that for example strengthens) of change.

Term used herein " upper sudden change " refers to that the Δ Δ G value of certain characteristic is better than the sudden change of parent's albumen (Δ Δ G value＜0).

Term used herein " lower sudden change " refers to the sudden change (Δ Δ G value＞0) of the Δ Δ G value difference of certain characteristic in parent's albumen.

Term used herein " productivity site " refers to given characteristic is had the position of sudden change at least one.

Term used herein " unproductive site " refers to given characteristic is not contained the position of upper sudden change.

Term " mutagenic primer " or " mutagenic oligonucleotide " (being used interchangeably in this article) are intended to represent corresponding to the part of template sequence and the oligonucleotide composition that can be hybrid with it. With regard to mutagenic primer, this primer generally not with template nucleic acid exact matching, the mispairing in primer is used for the sudden change that will expect introducing nucleic acid library. " non-mutagenic primer " used herein or " non-mutagenic oligonucleotide " refer to the oligonucleotide composition with template nucleic acid exact matching. In one embodiment of the invention, only use mutagenic primer. In another preferred embodiment of the present invention, primer is designed so that also to have comprised non-mutagenic primer in oligonucleotide mixture at least one zone that comprises mutagenic primer. By adding mutagenic primer and, corresponding to the mixture of the non-mutagenic primer of at least one mutagenic primer, likely producing the nucleic acid library that has multiple combination mutagenesis pattern. For example, if some members in expectation mutant nucleic acid library keep its precursor sequence in some position, and other members suddenly change at these places, site, and non-mutagenic primer provides the ability that obtains the not mutated body member of specified level in nucleic acid library for given residue. Method of the present invention is utilized mutagenesis and non-mutagenic oligonucleotide, and their general length is 10 to 50 bases, and more preferably length is about 15 to 45 bases. Yet, may be necessary to use less than 10 bases or the mutagenesis result that obtains to expect greater than the primer of 50 bases. For corresponding mutagenesis and non-mutagenic primer, not necessarily length is identical for corresponding oligonucleotides, but has overlapping in the zone of the sudden change corresponding to be added.

Can add primer with predetermined ratio according to the present invention. For example,, if certain specific sudden change of the level of signifiance and the difference sudden change of place, identical or different site small amount are contained in the library of expectation gained,, by the amount of primer that adjusting is added, likely produce the preference library of expectation. Perhaps,, by adding still less or the non-mutagenic primer of volume more, likely adjust the frequency that produces corresponding sudden change in the mutant nucleic acid library.

Term used herein " continuous mutation " refers to be present in the sudden change in same Oligonucleolide primers. For example, continuous mutation can adjoin each other or approach, yet they will be introduced in the mutant template nucleic acid of gained by same primer.

Term used herein " discontinuous sudden change " refers to be present in the sudden change in different Oligonucleolide primers. For example, discontinuous sudden change will be introduced in the mutant template nucleic acid of gained by the Oligonucleolide primers of preparation respectively.

Term " wild-type sequence " or " wild type gene " are used interchangeably in this article, refer to natural or naturally occurring sequence in host cell. In some embodiments, wild-type sequence refers to the purpose sequence as the starting point of protein engineering plan. The protein of wild-type sequence codified homology or allos. Homologous protein is that host cell need not to intervene the protein that namely produces. Allos albumen is that host cell is not intervened the protein that will not produce.

Term used herein " antibody " refers to immunoglobulin (Ig). Antibody includes but are not limited to the immunoglobulin (Ig) of any species that directly derive from expectation generation antibody. In addition, the present invention includes modification antibody. This term also refer to keep with complete antibody in conjunction with the antibody fragment of the ability of epi-position combination, and comprise polyclonal antibody, monoclonal antibody, chimeric antibody, antiidiotype (anti-ID) antibody. Antibody fragment includes but are not limited to complementarity-determining region (CDR), single-chain fragment variable region (scFv), variable region of heavy chain (VH), variable region of light chain (VL). Within polyclonal antibody and monoclonal antibody are also included within the present invention. Preferably, described antibody is monoclonal antibody.

Term " oxidation is stable " refers to protease of the present invention, and its common condition (as be exposed to or contact bleaching agent or oxidant) in proteolysis, hydrolysis, cleaning or other processes of the present invention lower maintenance keeps specific enzymatic activity amount after a period of time. In some embodiments, protease keeps at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after contact bleaching agent or oxidant a period of time (such as at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 16 minutes, 20 minutes etc.). In some embodiments, measure as described embodiments stability.

Term " chelating agent is stable " refers to protease of the present invention, and its common condition (as be exposed to or contact chelating agent) in proteolysis, hydrolysis, cleaning or other processes of the present invention is lower keeps after given a period of time keeping the enzymatic activity amount of appointment. In some embodiments, protease keeps at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after being exposed to given a period of time of chelating agent (such as at least 10 minutes, 20 minutes, 40 minutes, 60 minutes, 100 minutes etc.). In some embodiments, measure as described embodiments chelating agent stability.

Term " heat-staple " and " thermally-stabilised " refer to protease of the present invention, and it is exposed to assigned temperature under the common condition temperature of change (as be exposed to) and keeps the enzymatic activity amount of appointment after a period of time in proteolysis, hydrolysis, cleaning or other processes of the present invention. The temperature that changes comprises the temperature that raises or reduce. In some embodiments, protease keeps at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after temperature a period of time that is exposed to change (such as at least 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes etc.). In some embodiments, measure as described embodiments heat endurance.

Term " stability of enhancing " is compared as time goes by with other serine proteases (as subtilopeptidase A) and/or wild-type enzyme and the proteolytic activity that keeps is higher at the situation middle finger of oxidation, chelating agent, heat and/or pH stability protease.

Term " stability of reduction " is compared as time goes by with other serine proteases (as subtilopeptidase A) and/or wild-type enzyme and the proteolytic activity that keeps is lower at the situation middle finger of oxidation, chelating agent, heat and/or pH stability protease.

Term " cleaning is active " refers to protease obtains under common condition in proteolysis, hydrolysis, cleaning or other processes of the present invention clean-up performance. In some embodiments, clean-up performance measures to determine by applying the multiple cleaning that relates to enzyme sensitiveness spot (as the protein of grass, blood, breast or ovum), by spot being placed in after the standard wash condition, carrying out multiple chromatography, spectrophotometric or other quantitative approach and measures. Exemplary determination method includes but are not limited to those methods that the mensuration described in WO 99/34011 and United States Patent (USP) 6,605,458 (all incorporating this paper into as a reference) and embodiment comprise.

" the cleaning effective dose " of term protease refers to the amount of the above-mentioned protease of the enzymatic activity level of realization expectation in specific cleaning composition. Such effective dose is easy to be determined by those skilled in the art, and depend on many factors, apply, clean the concrete composition of composition and need still dry (as particle, bar shaped) composition of liquid composition etc. such as the concrete protease that uses, cleaning.

Term used herein " cleaning auxiliary material " refers to any liquid, solid or the gas material selected for the particular type of desirable cleaning composition and product form (for example liquid, particle, powder, bar shaped, pasty state, spraying, tablet, gel or foam composition), described material also preferably with said composition in the protease that uses compatible. In some embodiments, particulate composition is " densification " form, and in other embodiments, the liquid composition is " concentrating " form.

Term " performance of enhancing " is active in the cleaning to some enzyme sensitiveness spot (as ovum, breast, grass or blood) that the active situation middle finger of cleaning improves or increases, and by standard wash, circulate and/or the commonly used assessment of a plurality of washing after circulating determined.

Term " performance of reduction " is active in the cleaning to some enzyme sensitiveness spot (as ovum, breast, grass or blood) that the active situation middle finger of cleaning reduces or reduces, and the assessment commonly used after circulating by standard wash is determined.

Term " suitable performance " is at least 60% of the cleaning activity that cleans active situation middle finger subtilopeptidase A (as commercially available protease) relatively, at least 70%, at least 80%, at least 90 %, at least 95%, described subtilopeptidase A includes but are not limited to OPTIMASE^TMProtease (Genencor), PURAFECT^TMProtease product (Genencor), SAVINASE^TMProtease (Novozymes), BPN ' variant (consult as U.S. Patent application number 34,606), RELASE^TM、 DURAZYME ^TM、EVERLASE ^TM、KANNASE ^TMProtease (Novozymes), MAXACAL^TM、MAXAPEM ^TM、PROPERASE ^TMProtease (Genencor; Also consult U.S. Patent application numbers 34,606 and U.S. Patent number 5,700,676,5,955,340,6,312,936 and 6,482,628), and bacillus lentus misfolded proteins enzyme product (for example in WO 92/21760, WO 95/23221 and/or WO 97/07770 describe those). Exemplary Subtilisin protease variants includes but are not limited to and is being equivalent on the residue position of 76,101,103,104,120,159,167,170,194,195,217,232,235,236,245,248 and/or 252 of BPN ' have those that replace or lack. Clean-up performance can be by comparing protease of the present invention and those subtilopeptidase As to measure in multiple cleaning is measured, described cleaning is measured and is related to enzyme sensitiveness spot (as grass, blood or breast), measures by AAS or analytical method after the standard wash cycling condition.

" low washing agent concentration " used herein system comprises washing agent, wherein exists in washings lower than the about washing agent component of 800ppm. Japan's washing agent is considered to low washing agent concentration system usually, because they contain the washing agent component of the 667ppm that has an appointment usually in washings.

" middle washing agent concentration " used herein system comprises washing agent, wherein exists approximately 800ppm to the about washing agent component of 2000ppm in washings. The North America washing agent is considered to middle washing agent concentration system usually, because they contain the washing agent component of the 975ppm that has an appointment usually in washings. Brazil's washing agent contains the washing agent component of the 1500ppm that has an appointment usually in washings.

" high washing agent concentration " used herein system comprises washing agent, wherein exists in washings higher than the about washing agent component of 2000ppm. The Europe washing agent is considered to high washing agent concentration system usually, because they contain the washing agent component of the 3000-8000ppm that has an appointment usually in washings.

" fabric cleaning composition " used herein comprises the washing agent composition of hand-washing and machine-wash, and comprising laundry interpolation composition and the composition that is applicable to immersion and/or the stained fabric of pretreatment (as clothing, flax and other textile materials).

" non-woven cleaning composition " used herein comprises non-weaving (being fabric) surface cleaning composition, includes but are not limited to the washing agent composition that washes the dishes, oral cleaning composition, cleaning of teeth composition and personal cleaning compositions.

This paper cleans composition " densification " form preferably to be reflected by density, and the amount of composition by inorganic filling salt reflected. Inorganic filling salt is the conventional ingredient of powder type washing agent composition. In conventional washing agent composition, fill salt to measure and to exist significantly, be generally the 17-35% of composition weight. Compare, in fine and close composition, fill salt and exist with the amount that is no more than total composition 15%. In some embodiments, fill salt to be no more than composition weight 10%, or more preferably 5% amount exists. In some embodiments, inorganic filling salt is selected from sulfate and the hydrochloride of alkali and alkaline earth metal ions. Preferred filling salt is sodium sulphate.

II. serine protease of the present invention and sequence thereof

The invention provides the polynucleotides of separation, the amino acid sequence of its coding misfolded proteins enzyme. Complete this paper U.S. Patent application sequence number 10/576,331 as a reference of incorporating into provides the multiple protein enzyme, comprises wild type fiber monad serine protease and multiple misfolded proteins enzyme, signal coding sequence and amino acid sequence. In some preferred embodiments of the present invention, described fiber monad is fiber aeromonas strain 69B4 (DSM16035), described in U.S. Patent application sequence number 10/576,331.

A. serine protease

Although in given living species, the sequence of natural enzyme may be different, the enzyme of the particular type that the biology of same species produces generally under specified criteria the substrate specificity of (as temperature, pH, the water hardness, oxidizing condition, chelating condition and concentration) and/or proteolytic activity basic identical aspect horizontal. Therefore, with regard to purpose of the present invention, think that other bacterial strains of Cellulomonas and bacterial classification also produce fiber monad protease of the present invention, therefore for protease of the present invention, provide the source of use. In fact, as described herein, think that other members of micrococcus luteus suborder also can be used for the present invention.

In some embodiments, proteolysis polypeptide of the present invention characterizes aspect physical chemistry, and in other embodiments, they characterize based on its function, and in other embodiments, they use these two groups of characteristics to characterize simultaneously. The technology that the Physico-Chemical Characterization utilization is known, be used for measuring protein molecular weight such as SDS electrophoresis, gel filtration, amino acid composition, mass spectrum (such as MALDI-TOF-MS, LC-ES-MS/MS etc.) and sedimentation; Isoelectric focusing is used for measuring the pI of protein; Amino acid sequencing is used for measuring the amino acid sequence of protein; Crystallographic Study is used for measuring the tertiary structure of protein; Antibody is in conjunction with being used for measuring the antigenic epitopes that protein exists.

In some embodiments, functional characteristic is measured by the technology that protease field professional knows, and includes but are not limited to the multiple business substrate of hydrolysis, as dimethyl casein (" DMC ") and/or AAPF-pNA. The preferred technology of this functional sign is described in greater detail in the embodiment of this paper.

Ripe protease also shows the proteolytic activity hydrolysing activity of the substrate (as DMC) that contains peptide bond (for example to). In other embodiments, protease of the present invention provides the scourability of enhancing under specified requirements. Although the present invention includes protease 69B as herein described, but in some embodiments, protease demonstration of the present invention is in a ratio of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity with the 69B4 proteolytic activity. In some embodiments, described protease with trade name

(Novzymes) or

The proteolytic activity of the protease of (Genencor) selling is compared and is shown under the same conditions at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity. In some embodiments, protease of the present invention is being compared demonstration quite or the scourability of enhancing with the 69B4 under the same terms under the condition of appointment. In some preferred embodiments, protease of the present invention is under the condition of appointment and with trade name

(Novzymes) or

(Genencor) protease of selling is compared the scourability that shows quite or strengthen under the same conditions.

In other embodiments, the polynucleotides of protease of the present invention and/or encoding said proteins enzyme with the form of purifying (namely, exist with the concentration higher or lower than in natural or wild-type biology in specific composition) provide, perhaps with the rear common non-existent component combination of expression in natural or wild-type biology. Yet the present invention is not intended to the protease that is subject to any specific purity level, because the purity level of protease can be used for being fit to the multiple application of protease of the present invention.

B. the nucleic acid of serine protease and amino acid sequence

DNA sequence (SEQ ID NO:1) from the asp gene of fiber aeromonas strain 69B4 (DSM 16035) hereinafter is provided. The initial polynucleotides of the signal peptide of coding fiber aeromonas strain 69B4 protease are runic (ATG). This sequence also comprises signal peptide and precursor serine protease.

Following DNA sequence dna (SEQ ID NO:2) coding and the effective signal peptide (SEQ ID NO:9) that is connected of the precursor protein enzyme (SEQ ID NO:7) from fiber aeromonas strain 69B4 (DSM 16035). The initial polynucleotides of the signal peptide of coding fiber aeromonas strain 69B4 protease are runic (ATG). Terminator codon (TGA) starts from 1486 residues. 85,595 and 1162 residues relate separately to the initial residue of N end former sequence, mature sequence and the former sequence of c-terminus, with runic, show and are added with underscore.

ATGACACCAC GCACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTG 60

GCAGGCGGCA TGGCCGCCCA GGCCAACGAG CCCGCACCAC CCGGGAGCGC GAGCGCACCG 120

CCACGCCTGG CCGAGAAGCT CGACCCCGAC CTCCTCGAGG CCATGGAGCG CGACCTGGGC 180

CTCGACGCGG AGGAAGCCGC CGCCACCCTG GCGTTCCAGC ACGACGCAGC CGAGACCGGC 240

GAGGCCCTCG CCGAAGAGCT CGACGAGGAC TTCGCCGGCA CCTGGGTCGA GGACGACGTC 300

CTGTACGTCG CCACCACCGA CGAGGACGCC GTCGAGGAGG TCGAGGGCGA AGGCGCCACG 360

GCCGTCACCG TCGAGCACTC CCTGGCCGAC CTCGAGGCCT GGAAGACCGT CCTCGACGCC 420

GCCCTCGAGG GCCACGACGA CGTGCCCACC TGGTACGTCG ACGTCCCGAC CAACAGCGTC 480

GTCGTCGCCG TCAAGGCCGG AGCCCAGGAC GTCGCCGCCG GCCTCGTCGA AGGTGCCGAC 540

GTCCCGTCCG ACGCCGTGAC CTTCGTCGAG ACCGACGAGA CCCCGCGGAC CATGTTCGAC 600

GTGATCGGCG GCAACGCCTA CACCATCGGG GGGCGCAGCC GCTGCTCGAT CGGGTTCGCG 660

GTCAACGGCG GGTTCATCAC CGCCGGCCAC TGCGGCCGCA CCGGCGCCAC CACCGCCAAC 720

CCCACCGGGA CCTTCGCCGG GTCCAGCTTC CCGGGCAACG ACTACGCGTT CGTCCGTACC 780

GGGGCCGGCG TGAACCTGCT GGCCCAGGTC AACAACTACT CCGGTGGCCG CGTCCAGGTC 840

GCCGGGCACA CCGCGGCCCC CGTCGGCTCG GCCGTGTGCC GGTCCGGGTC GACCACCGGG 900

TGGCACTGCG GCACCATCAC TGCGCTCAAC TCCTCGGTCA CCTACCCCGA GGGCACCGTC 960

CGCGGCCTGA TCCGCACCAC CGTCTGCGCC GAGCCCGGCG ACTCCGGTGG CTCGCTGCTC 1020

GCCGGCAACC AGGCCCAGGG CGTCACGTCC GGCGGCTCCG GCAACTGCCG CACCGGTGGC 1080

ACCACGTTCT TCCAGCCGGT CAACCCCATC CTCCAGGCGT ACGGCCTGAG GATGATCACC 1140

ACGGACTCGG GCAGCAGCCC GGCCCCTGCA CCGACCTCCT GCACCGGCTA CGCCCGCACC 1200

TTCACCGGGA CCCTCGCGGC CGGCCGGGCC GCCGCCCAGC CCAACGGGTC CTACGTGCAG 1260

GTCAACCGGT CCGGGACCCA CAGCGTGTGC CTCAACGGGC CCTCCGGTGC GGACTTCGAC 1320

CTCTACGTGC AGCGCTGGAA CGGCAGCTCC TGGGTGACCG TCGCCCAGAG CACCTCCCCC 1380

GGCTCCAACG AGACCATCAC CTACCGCGGC AACGCCGGCT ACTACCGCTA CGTGGTCAAC 1440

GCCGCGTCCG GCTCCGGTGC CTACACCATG GGGCTCACCC TCCCCTGA 1488

(SEQ ID NO：2)

Following DNA sequence dna (SEQ ID NO:3) coding is from the precursor protein enzyme of fiber aeromonas strain 69B4 (DSM 16035).

1 AACGAGCCCG CACCACCCGG GAGCGCGAGC GCACCGCCAC GCCTGGCCGA GAAGCTCGAC

TTGCTCGGGC GTGGTGGGCC CTCGCGCTCG CGTGGCGGTG CGGACCGGCT CTTCGAGCTG

61 CCCGACCTCC TCGAGGCCAT GGAGCGCGAC CTGGGCCTCG ACGCGGAGGA AGCCGCCGCC

GGGCTGGAGG AGCTCCGGTA CCTCGCGCTG GACCCGGAGC TGCGCCTCCT TCGGCGGCGG

121 ACCCTGGCGT TCCAGCACGA CGCAGCCGAG ACCGGCGAGG CCCTCGCCGA AGAGCTCGAC

TGGGACCGCA AGGTCGTGCT GCGTCGGCTC TGGCCGCTCC GGGAGCGGCT TCTCGAGCTG

181 GAGGACTTCG CCGGCACCTG GGTCGAGGAC GACGTCCTGT ACGTCGCCAC CACCGACGAG

CTCCTGAAGC GGCCGTGGAC CCAGCTCCTG CTGCAGGACA TGCAGCGGTG GTGGCTGCTC

241 GACGCCGTCG AGGAGGTCGA GGGCGAAGGC GCCACGGCCG TCACCGTCGA GCACTCCCTG

CTGCGGCAGC TCCTCCAGCT CCCGCTTCCG CGGTGCCGGC AGTGGCAGCT CGTGAGGGAC

301 GCCGACCTCG AGGCCTGGAA GACCGTCCTC GACGCCGCCC TCGAGGGCCA CGACGACGTG

CGGCTGGAGC TCCGGACCTT CTGGCAGGAG CTGCGGCGGG AGCTCCCGGT GCTGCTGCAC

361 CCCACCTGGT ACGTCGACGT CCCGACCAAC AGCGTCGTCG TCGCCGTCAA GGCCGGAGCC

GGGTGGACCA TGCAGCTGCA GGGCTGGTTG TCGCAGCAGC AGCGGCAGTT CCGGCCTCGG

421 CAGGACGTCG CCGCCGGCCT CGTCGAAGGT GCCGACGTCC CGTCCGACGC CGTGACCTTC

GTCCTGCAGC GGCGGCCGGA GCAGCTTCCA CGGCTGCAGG GCAGGCTGCG GCACTGGAAG

481 GTCGAGACCG ACGAGACCCC GCGGACCATG TTCGACGTGA TCGGCGGCAA CGCCTACACC

CAGCTCTGGC TGCTCTGGGG CGCCTGGTAC AAGCTGCACT AGCCGCCGTT GCGGATGTGG

541 ATCGGGGGGC GCAGCCGCTG CTCGATCGGG TTCGCGGTCA ACGGCGGGTT CATCACCGCC

TAGCCCCCCG CGTCGGCGAC GAGCTAGCCC AAGCGCCAGT TGCCGCCCAA GTAGTGGCGG

601 GGCCACTGCG GCCGCACCGG CGCCACCACC GCCAACCCCA CCGGGACCTT CGCCGGGTCC

CCGGTGACGC CGGCGTGGCC GCGGTGGTGG CGGTTGGGGT GGCCCTGGAA GCGGCCCAGG

661 AGCTTCCCGG GCAACGACTA CGCGTTCGTC CGTACCGGGG CCGGCGTGAA CCTGCTGGCC

TCGAAGGGCC CGTTGCTGAT GCGCAAGCAG GCATGGCCCC GGCCGCACTT GGACGACCGG

721 CAGGTCAACA ACTACTCCGG TGGCCGCGTC CAGGTCGCCG GGCACACCGC GGCCCCCGTC

GTCCAGTTGT TGATGAGGCC ACCGGCGCAG GTCCAGCGGC CCGTGTGGCG CCGGGGGCAG

781 GGCTCGGCCG TGTGCCGGTC CGGGTCGACC ACCGGGTGGC ACTGCGGCAC CATCACTGCG

CCGAGCCGGC ACACGGCCAG GCCCAGCTGG TGGCCCACCG TGACGCCGTG GTAGTGACGC

841 CTCAACTCCT CGGTCACCTA CCCCGAGGGC ACCGTCCGCG GCCTGATCCG CACCACCGTC

GAGTTGAGGA GCCAGTGGAT GGGGCTCCCG TGGCAGGCGC CGGACTAGGC GTGGTGGCAG

901 TGCGCCGAGC CCGGCGACTC CGGTGGCTCG CTGCTCGCCG GCAACCAGGC CCAGGGCGTC

ACGCGGCTCG GGCCGCTGAG GCCACCGAGC GACGAGCGGC CGTTGGTCCG GGTCCCGCAG

961 ACGTCCGGCG GCTCCGGCAA CTGCCGCACC GGTGGCACCA CGTTCTTCCA GCCGGTCAAC

TGCAGGCCGC CGAGGCCGTT GACGGCGTGG CCACCGTGGT GCAAGAAGGT CGGCCAGTTG

1021 CCCATCCTCC AGGCGTACGG CCTGAGGATG ATCACCACGG ACTCGGGCAG CAGCCCGGCC

GGGTAGGAGG TCCGCATGCC GGACTCCTAC TAGTGGTGCC TGAGCCCGTC GTCGGGCCGG

1081 CCTGCACCGA CCTCCTGCAC CGGCTACGCC CGCACCTTCA CCGGGACCCT CGCGGCCGGC

GGACGTGGCT GGAGGACGTG GCCGATGCGG GCGTGGAAGT GGCCCTGGGA GCGCCGGCCG

1141 CGGGCCGCCG CCCAGCCCAA CGGGTCCTAC GTGCAGGTCA ACCGGTCCGG GACCCACAGC

GCCCGGCGGC GGGTCGGGTT GCCCAGGATG CACGTCCAGT TGGCCAGGCC CTGGGTGTCG

1201 GTGTGCCTCA ACGGGCCCTC CGGTGCGGAC TTCGACCTCT ACGTGCAGCG CTGGAACGGC

CACACGGAGT TGCCCGGGAG GCCACGCCTG AAGCTGGAGA TGCACGTCGC GACCTTGCCG

1261 AGCTCCTGGG TGACCGTCGC CCAGAGCACC TCCCCCGGCT CCAACGAGAC CATCACCTAC

TCGAGGACCC ACTGGCAGCG GGTCTCGTGG AGGGGGCCGA GGTTGCTCTG GTAGTGGATG

1321 CGCGGCAACG CCGGCTACTA CCGCTACGTG GTCAACGCCG CGTCCGGCTC CGGTGCCTAC

GCGCCGTTGC GGCCGATGAT GGCGATGCAC CAGTTGCGGC GCAGGCCGAG GCCACGGATG

1381 ACCATGGGGC TCACCCTCCC CTGA(SEQID NO：3)

TGGTACCCCG AGTGGGAGGG GACT

Following DNA sequence dna (SEQ ID NO:4) coding is from the maturation protein enzyme of fiber aeromonas strain 69B4 (DSM 16035).

1 TTCGACGTGA TCGGCGGCAA CGCCTACACC ATCGGGGGGC GCAGCCGCTG CTCGATCGGG

AAGCTGCACT AGCCGCCGTT GCGGATGTGG TAGCCCCCCG CGTCGGCGAC GAGCTAGCCC

61 TTCGCGGTCA ACGGCGGGTT CATCACCGCC GGCCACTGCG GCCGCACCGG CGCCACCACC

AAGCGCCAGT TGCCGCCCAA GTAGTGGCGG CCGGTGACGC CGGCGTGGCC GCGGTGGTGG

121 GCCAACCCCA CCGGGACCTT CGCCGGGTCC AGCTTCCCGG GCAACGACTA CGCGTTCGTC

CGGTTGGGGT GGCCCTGGAA GCGGCCCAGG TCGAAGGGCC CGTTGCTGAT GCGCAAGCAG

181 CGTACCGGGG CCGGCGTGAA CCTGCTGGCC CAGGTCAACA ACTACTCCGG TGGCCGCGTC

GCATGGCCCC GGCCGCACTT GGACGACCGG GTCCAGTTGT TGATGAGGCC ACCGGCGCAG

241 CAGGTCGCCG GGCACACCGC GGCCCCCGTC GGCTCGGCCG TGTGCCGGTC CGGGTCGACC

GTCCAGCGGC CCGTGTGGCG CCGGGGGCAG CCGAGCCGGC ACACGGCCAG GCCCAGCTGG

301 ACCGGGTGGC ACTGCGGCAC CATCACTGCG CTCAACTCCT CGGTCACCTA CCCCGAGGGC

TGGCCCACCG TGACGCCGTG GTAGTGACGC GAGTTGAGGA GCCAGTGGAT GGGGCTCCCG

361 ACCGTCCGCG GCCTGATCCG CACCACCGTC TGCGCCGAGC CCGGCGACTC CGGTGGCTCG

TGGCAGGCGC CGGACTAGGC GTGGTGGCAG ACGCGGCTCG GGCCGCTGAG GCCACCGAGC

421 CTGCTCGCCG GCAACCAGGC CCAGGGCGTC ACGTCCGGCG GCTCCGGCAA CTGCCGCACC

GACGAGCGGC CGTTGGTCCG GGTCCCGCAG TGCAGGCCGC CGAGGCCGTT GACGGCGTGG

481 GGTGGCACCA CGTTCTTCCA GCCGGTCAAC CCCATCCTCC AGGCGTACGG CCTGAGGATG

CCACCGTGGT GCAAGAAGGT CGGCCAGTTG GGGTAGGAGG TCCGCATGCC GGACTCCTAC

561 ATCACCACGG ACTCGGGCAG CAGCCCG(SEQ ID NO：4)

TAGTGGTGCC TGAGCCCGTC GTCGGGC

Following DNA sequence dna (SEQ ID NO:5) coding is from the signal peptide of fiber aeromonas strain 69B4 (DSM 16035).

1 ATGACACCAC CACAGTCAC GCGGGCCCTG GCCGTGGCCA CCGCAGCCGC CACACTCCTG

TACTGTGGTG CGTGTCAGTG CGCCCGGGAC CGGCACCGGT GGCGTCGGCG GTGTGAGGAC

61 GCAGGCGGCA TGGCCGCCCA GGCC(SEQ ID NO：5)

CGTCCGCCGT ACCGGCGGGT CCGG

Following sequence is from the burst of fiber aeromonas strain 69B4 (DSM 16035) and the amino acid sequence of precursor protein enzyme (SEQ ID NO:6), it comprises burst [1a-c section] (1-28 position residue [198 to-171]), N end former sequence [2a-r section] (29-198 position residue [170 to-1]), maturation protein enzyme [3a-t section] (199-387 position residue [1-189]) and C end former sequence [4a-l section] (388-495 position residue [190-398]), by DNA sequence encoding shown in SEQ ID NOS:1,2,3 and 4. The N terminal sequence of maturation protein enzyme amino acid sequence is runic.

1 MTPRTVTRAL AVATAAATLL AGGMAAQA NE PAPPGSASAP PRLAEKLDPD

1a 1b 1c 2a 2b 2c

51 LLEAMERDLG LDAEEAAATL AFQHDAAETG EALAEELDED FAGTWVEDDV

2d 2e 2f 2g 2h

101 LYVATTDEDA VEEVEGEGAT AVTVEHSLAD LEAWKTVLDA ALEGHDDVPT

2i 2j 2k 21 2m

151 WYVDVPTNSV VVAVKAGAQD VAAGLVEGAD VPSDAVTFVE TDETPRTM FD

2n 2o 2p 2q 2r 3a

201 VIGGNAYTIG GRSRCSIGFA VNGGFITAGH CGRTGATTAN PTGTFAGSSF

3b 3c 3d 3e 3f

251 PGNDYAFVRT GAGVNLLAQV NNYSGGRVQV AGHTAAPVGS AVCRSGSTTG

3g 3h 3i 3j 3k

301 WHCGTITALN SSVTYPEGTV RGLIRTTVCA EPGDSGGSLL AGNQAQGVTS

31 3m 3n 3o 3p

351 GGSGNCRTGG TTFFQPVNPI LQAYGLRMIT TDSGSSP APA PTSCTGYART

3q 3r 3s 3t 4a4b

401 FTGTLAAGRA AAQPNGSYVQ VNRSGTHSVC LNGPSGADFD LYVQRWNGSS

4c 4d 4e 4f 4g

451 WVTVAQSTSP GSNETITYRG NAGYYRYVVN AASGSGAYTM GLTLP (SEQ ID NO：6)

4h 4i 4j 4k 4l

Following sequence (SEQ ID NO:7) is the amino acid sequence (SEQ ID NO:7) from the precursor protein enzyme of fiber aeromonas strain 69B4 (DSM 16035).

1 NEPAPPGSAS APPRLAEKLD PDLLEAMERD.LGLDAEEAAA.TLAFQHDAAE

51 TGEALAEELD EDFAGTWVED DVLYVATTDE DAVEEVEGEG ATAVTVEHSL

101 ADLEAWKTVL DAALEGHDDV PTWYVDVPTN SVVVAVKAGA QDVAAGLVEG

151 ADVPSDAVTF VETDETPRTM FDVIGGNAYT IGGRSRCSIG FAVNGGFITA

201 GHCGRTGATT ANPTGTFAGS SFPGNDYAFV RTGAGVNLLA QVNNYSGGRV

251 QVAGHTAAPV GSAVCRSGST TGWHCGTITA LNSSVTYPEG TVRGLIRTTV

301 CAEPGDSGGS LLAGNQAQGV TSGGSGNCRT GGTTFFQPVN PILQAYGLRM

351 ITTDSGSSPA PAPTSCTGYA RTFTGTLAAG RAAAQPNGSY VQVNRSGTHS

401 VCLNGPSGAD FDLYVQRWNG SSWVTVAQST SPGSNETTTY RGNAGYYRYV

451 VNAASGSGAY TMGLTLP(SEQ ID NO：7)

Following sequence (SEQ ID NO:8) is the amino acid sequence from the maturation protein enzyme of fiber aeromonas strain 69B4 (DSM 16035). Catalytic triplet residue H32, D56 and S132 are runic and are added with underscore.

1 FDVIGGNAYT IGGRSRCSIG FAVNGGFITA G HCGRTGATT ANPTGTFAGS

51 SFPGN DYAFV RTGAGVNLLA QVNNYSGGRV QVAGHTAAPV GSAVCRSGST

101 TGWHCGTITA LNSSVTYPEG TVRGLIRTTV CAEPGD SGGS LLAGNQAQGV

151 TSGGSGNCRT GGTTFFQPVN PILQAYGLRM ITTDSGSSP(SEQ ID NO：8)

Following sequence (SEQ ID NO:9) is the amino acid sequence from the signal peptide of the protease of fiber aeromonas strain 69B4 (DSM 16035).

1 MTPRTVTRAL AVATAAATLL AGGMAAQA(SEQ ID NO：9)

In some embodiments, the present invention includes variant, it comprises at least shown in SEQ ID NO:1 in polynucleotides length is the about part of 1621 base-pairs. In some particularly preferred embodiments, the invention provides with wild type (as " parent ") serine protease and compare the variant with multiple mutation. At some these more in particularly preferred embodiment, the variant of these multiple mutations is compared with wild type (for example " parent ") serine protease and is shown improved performance.

it will be understood by those skilled in the art that, degeneracy due to genetic code, can encode this paper and/or as with reference to the whole U.S. Patent application series number 10/576 of incorporating this paper into of multiple polynucleotides, the signal peptide that provides in 331, precursor protein enzyme and/or maturation protein enzyme (for example SEQ ID NO:6,7 and/or 8 of U.S. Patent application series number 10/576,331) or the protease with above-mentioned percentage sequence homogeneity. another embodiment of the present invention comprises polynucleotides, it comprises and difference code signal peptide and precursor protein enzyme, the SEQ ID NO:2 of precursor protein enzyme and/or maturation protein enzyme, 3 and/or 4 polynucleotide sequence has respectively at least 70% sequence homogeneity, at least 75% sequence homogeneity, at least 80% sequence homogeneity, at least 85% sequence homogeneity, at least 90% sequence homogeneity, at least 92% sequence homogeneity, at least 95% sequence homogeneity, at least 97% sequence homogeneity, the nucleotide sequence of at least 98% sequence homogeneity and at least 99% sequence homogeneity.

in other embodiments, the invention provides fragment or the part of the DNA of encoding proteins enzyme, as long as the fragment of its coding keeps proteolytic activity. another embodiment of the present invention comprises at least 20% sequence length in the SEQ ID NO:1 polynucleotide sequence with coding precursor protein enzyme, at least 30% sequence length, at least 40% sequence length, at least 50% sequence length, at least 60% sequence length, 70% sequence length, at least 75% sequence length, at least 80% sequence length, at least 85% sequence length, at least 90% sequence length, at least 92% sequence length, at least 95% sequence length, at least 97% sequence length, the polynucleotides of at least 98% sequence length and at least 99% sequence. in alternative embodiment, these fragments of described sequence length or part are the continuous part of described sequence length, are used for the reorganization (for example seeing U.S. Patent number 6,132,970) at recombinant DNA sequence DNA sequence dna.

Another embodiment of the present invention comprises the fragment of DNA described herein, it can be used for obtaining the DNA fragmentation of partial-length according to technology known in the art, described fragment can be used for separating or identification code described herein from the maturation protein enzyme of fiber monad 69B4 or the polynucleotides of its fragment with proteolytic activity. In addition, the DNA that provides in SEQ ID NO:1 can be used for identifying from the encoding proteins enzyme of other species (particularly from the fiber monad) or the homologous dna fragment of its part with proteolytic activity.

In addition, the present invention includes and use the primer that built by SEQ ID NO:1 or its suitable part or fragment (for example at least about 5 to 20 or 10 to 15 continuous nucleotides) or probe sequence probe or the primer as the nucleic acid in screening genome or cDNA source. In some embodiments, the invention provides the DNA probe with expectation length (that is, general length be 100 to 1000 bases) based on sequence in SEQ ID NO:1.

In some embodiments,, by the described DNA fragmentation of electrophoretic separation, downcut from gel, and reclaim from the agar matrix of gel. In preferred embodiments, the DNA fragmentation of following this purifying carries out mark (for example according to manufacturer's explanation, using the Megaprime Mk system) to mix P in DNA³². 95 ℃ of given times (as 5 minutes) of heating make probe sex change through mark, and add immediately in film and prehybridization solution. Hybridization reaction carry out under suitable condition reasonable time (as 37 ℃ 18 hours), and softly shake or rotate. Rinsing film (for example washing in SSC/0.3%SDS twice), then with suitable washing solution washing and soft the stirring. The stringency of expectation is the reflection of washing film (filter) condition used. In some embodiments of this paper, " low strict " condition is included under 20 ℃ and washed 15 minutes in the solution of 0.2 * SSC/0.1%SDS; And in other embodiments, " in strict " condition is included under 37 ℃ another washing step of 30 minutes of washing in the solution of 0.2 * SSC/0.1%SDS; In other embodiments, " high strict " condition is included under 37 ℃ and washs another washing step of 45 minutes in the solution of 0.2 * SSC/0.1%SDS; In other embodiments, " the highest strict " condition is included under 37 ℃ and washs another washing step of 60 minutes in the solution of 0.2 * SSC/0.1%SDS. Therefore, a plurality of embodiment of the present invention provide can be under middle and high and/or the highest stringent condition with from SEQ ID NO:1 or 2 polynucleotides that the Probe Hybridization of nucleotide sequence is provided.

After washing, that film is dry and detect the probe of combination. If use P³²Or other radio isotopes reagent that serves as a mark, by autoradiograph, detect the probe of combination. The other technologies that other probes are manifested are well known to those skilled in the art. Within the detection of bonding probes (its expression nucleotide sequence has the homology of expectation with any purpose sequence that this paper provides, so also has homogeneity) is included in the present invention. Therefore, the invention provides the method that detects the code book nucleic acid of protease that invention comprises, described nucleic acid comprises in SEQ ID NO:1 nucleotide sequence part or all of with other nucleic acid hybridizations in genome or cDNA source.

As described above, in other embodiments, the hybridization condition is based on the melting temperature (Tm) of nucleic acid in conjunction with complex, to give " stringency " determined hereinafter explained. " the highest stringency " generally is present in approximately Tm-5 ℃ (following 5 ℃ of probe Tm); " high stringency " is following approximately 5 ℃ to 10 ℃ of Tm; " middle stringency " is following approximately 10 ℃ to 20 ℃ of Tm; " low stringency " is following approximately 20 ℃ to 25 ℃ of Tm. As is known to the person skilled in the art, select middle and high and/or the highest stringency hybridization, with the polynucleotide sequence that condition is optimized for evaluation or detection and polynucleotide sequence homology or is equal to.

In other embodiments, the invention provides the nucleic acid construct (being expression vector) that comprises nucleotides more than code book invention protease. In other embodiments, the invention provides and transform the host cell that at least a these carriers are arranged.

In other embodiments, the invention provides the polynucleotide sequence of going back coded signal sequence, as described in the U.S. Patent application series number 10/576,331 as incorporate this paper into reference to integral body. In some these embodiments, the invention provides the sequence with the burst of inferring, and can be under middle and high and/or the highest stringency condition and the polynucleotides of Probe Hybridization from the described nucleotide sequence of SEQ ID NO:1, it is active that wherein said burst has the essentially identical signal of the burst of with polynucleotides of the present invention, encoding.

In some embodiments, described signal activity is secreted in fermentation medium and represents with essentially identical level by protease and parent material, and is as described in U.S. Patent application series number 10/576,331. In the mensuration gram positive host cell, the additive method of the secretion level of allos or homologous protein and detection secretory protein comprises using that this albumen is had specific polyclone or monoclonal antibody. Example comprises enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA) and fluorescence-activated cell sorting (FACS), as well known to the skilled person.

other aspects of the present invention comprise and this paper and U.S. Patent application series number 10/576, 331 described SEQ ID NOS:7 or 8 amino acid sequence comprise 65% amino acid sequence identity, at least 70% sequence homogeneity, at least 75% amino acid sequence identity, at least 80% amino acid sequence identity, at least 85% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 95% amino acid sequence identity, at least 97% amino acid sequence identity, at least 98% amino acid sequence identity and at least 99% amino acid sequence identity have a proteolytic activity polypeptide. the proteolytic activity of these polypeptide is measured with means known in the art, comprises the method for assessment of the washing agent function. in other embodiments, described polypeptide separates.

III. obtain the polynucleotides of code book invention micrococcus luteus (for example fiber monad) protease

In some embodiments, obtained the nucleic acid of code book invention protease by for example cloned DNA (as DNA " library "), chemical synthesis, cDNA clone, PCR, cloned genomic dna or its fragment by standard method known in the art, perhaps purifying (is consulted as Sambrook etc., the same [1989] from the cell (as bacterium and fungi species) of expectation; And Glover and Hames edit,DNA Cloning：A Practical Approach, the 1st and 2 volumes, second edition). Synthesizing of polynucleotide sequence is known in the art (consults as Beaucage and Caruthers, Tetrahedron Lett., 22:1859-1862[1981]), comprise and use the automation synthesizer (to consult as Needham-VanDevanter etc., Nucl.Acids Res., 12:6159-6168[1984]). The DNA sequence can also customize, and from a plurality of business source, orders. As described in more detail, in some embodiments, from the nucleotide sequence of genomic DNA, contain regulatory region and code area.

In generation some embodiments from the molecular cloning of the gene of genomic DNA, produce DNA fragmentation, some of them comprise at least a portion of desired gene. In some embodiments, use multiple Restriction Enzyme at the specific site cutting DNA. In some alternative embodiment, under existing, manganese carrys out DNA breakage with the DNA enzyme, and perhaps physics is sheared DNA (for example by ultrasonic). Follow and use standard technique according to size separation linear DNA fragment and amplification, including but are not limited to agarose gel electrophoresis, PCR and column chromatography.

In case produced nucleic acid fragment, just the specific DNA fragment of identification code protease in many ways. For example, in some embodiments, with the asp gene of encoding proteins hydrolase or its specific RNA or its fragment (as probe or primer) separate, mark, being then used in hybridization well known in the art measures, with the gene for detection of producing (consult as Benton and Davis Science 196:180[1977]; And Grunstein and Hogness, Proc.Natl.Acad.Sci.USA 72:3961[1975]). In preferred embodiments, with the DNA fragmentation of the total significant sequence similarity of probe in hybridize under paramount stringent condition.

In some preferred embodiments, use as known in the art PCR to increase. In some preferred embodiments, arrive up to about 60 nucleotides, preferred approximately 12 to 30 nucleotides at least about 4 nucleotides with any suitable being used in combination in SEQ ID NO:1-5, more preferably from about the nucleotide sequence of 25 nucleotides (as fragment) is as the PCR primer. These identical fragments also can be used as the probe in hybridization and product detection method.

In some embodiments, utilize the PCR that carries out with degeneracy oligonucleotide primer while separating nucleic acid construct of the present invention from cDNA or genomic library, described primer prepares based on the amino acid sequence of this protein. Primer can be any section length, and for example length is at least 4, at least 5, at least 8, at least 15, at least 20 nucleotides.

In view of above content, should be appreciated that, the polynucleotide sequence of the polynucleotide sequence based on SEQ ID NO:1-5 provided herein can be used for from other species (particularly bacterium) obtaining the polynucleotide sequence of identical or homology, and its coding has the enzyme of the serine protease that protease 69B4 shows. Other sequences are provided in U.S. Patent application series number 10/576,331.

IV. the multiple mutation variants of serine protease of the present invention

As described herein, in particularly preferred embodiments, the invention provides the multiple mutation variants of serine protease. In some the most preferred embodiment, these variants are compared with parent's (as wild type) protease and are shown improved performance. In some these the most preferred embodiment, described variant has improved scourability, LAS stability and/or proteolytic activity. Therefore, these variants can be used for multiple application, include but are not limited to the washing agent that washes the dishes, laundry washing agent and cleaning surfaces washing agent.

V. express and reclaim serine protease of the present invention

Any appropriate method for expression and recovery serine protease of the present invention all can be used for herein. In fact, those skilled in the art understand the many methods that are suitable for cloning from the polypeptide with proteolytic activity and other enzymes (the another kind of peptide that for example has proteolytic activity, as protease, cellulase, mannase or amylase etc.) of fiber monad. This area also becomes known at least one of the polynucleotides of code book invention enzyme (for example a plurality of) copy and any other required sequence are combined and introduce many methods in host cell gene or genome.

Generally speaking, be used for clone gene and to the standard method that described gene is introduced foreign protein enzyme code area (a plurality of copies that comprise the external source code area), can be used for obtaining Cellulomonas 69B4 protease derivative or its homologue. In fact, this specification (comprising embodiment) provides such instruction. Yet additive method known in the art is also (consulting as Sambrook etc., the same (1989) of being suitable for; Ausubel etc., the same [1995]; And Harwood and Cutting editMolecular Biological Methods for Bacillus，" John Wiley and Sons, [1990]; With WO 96/34946).

In some preferred embodiments, polynucleotide sequence of the present invention is by it effectively is connected to express in suitable expression vector with expression control sequenc, and the technology of use this area maturation transforms suitable host with this expression vector. In some embodiments, according to technology ripe in this area polypeptide that separation and purifying produce after expression DNA sequence dna of the present invention from the fermentate of cell culture in many ways. Those skilled in the art can select most suitable separation and purification technique.

More specifically, the invention provides construct, the carrier that comprises polynucleotides described herein; Host cell with these carriers conversions; The protease of these host cell expressions; Expression and system for generation of the serine protease from microorganism (particularly micrococcus luteus suborder member, include but are not limited to fiber monad species). In some embodiments, produce with the polynucleotides of encoding serine protease the restructuring host cell that is suitable for expressing this serine protease. In some preferred embodiments, expressive host can be with business volume production raw albumen enzyme. Other details are provided in U.S. Patent application series number 10/576,331.

VI. recombinant vector and host cell

As described above, in some embodiments, the invention provides the carrier that comprises above-mentioned polynucleotides. In some embodiments, the carrier of the present invention of encoding proteins enzyme (being construct) from the genome source (for example, the preparation of use genomic library, and, according to standard technique, hybridize to screen all or part of DNA sequence dna of encoding proteins enzyme with synthetic oligonucleotide probe). These carriers are described in greater detail in U.S. Patent application series number 10/576,331.

As above-mentioned, in some embodiments, the present invention also provides the host cell that transforms with above-mentioned carrier. Other host cells are described in greater detail in U.S. Patent application series number 10/576,331.

VII. the application of serine protease

As described in more detail, protease of the present invention has and makes it be highly suitable for the key character of some application. For example, protease of the present invention has the heat endurance of enhancing. In some embodiments, described enzyme is compared the oxidation stability of also demonstration enhancing and the chelating agent stability of enhancing with some protease of present use. Therefore, these protease can be used for cleaning in composition. In fact, under some washing condition, protease of the present invention demonstrate with the subtilopeptidase A of present use quite or the scourability that strengthens. Therefore, cleaning of the present invention and/or enzyme composition are considered to provide with the form of multiple cleaning composition. In some embodiments, protease of the present invention uses in the mode identical with subtilopeptidase A (protease that namely uses at present). Therefore, the present invention can be used for multiple cleaning composition and animal feed, leather processing (as softening), proteolysis and textile applications. Described protease also can be used for the personal hygiene application.

Therefore, protease of the present invention can be used for multiple commercial Application, particularly cleans, sterilization, animal feed and weaving/leather industry. In some embodiments, protease of the present invention and washing agent, buider, bleaching agent and the combination of other conventional ingredients, to produce multiple new cleaning composition, they can be used for laundry and other cleaning fields, the washing agent of for example doing washing (powder with liquid), laundry preimpregnation infusion, full fabric bleaching agent, wash the dishes application and the pipe-dredging agent of washing agent (liquid with powder), household cleaners, particularly strip soap and liquid soap automatically. In addition, described protease can be used for cleaning contact lenses and other article, and this realizes by the aqueous solution that makes these materials contact this cleaning composition. In addition, these natural protease can be used for such as hydrolase polypeptide, refuse processing, textile application, Medical Devices cleaning, biomembrane is removed and in protein production as merging nickase etc. The composition of these products is not critical for the purpose of the present invention, as long as protease keeps its function in environment used. In some embodiments, the combined composition that easily prepares of conventional component of the said composition of the protease by will clean effective dose or the enzyme composition that comprises this protease preparation and amount known in this field.

Protease of the present invention can be used for cleaned industry especially, includes but are not limited to laundry and the washing agent that washes the dishes. These application are placed in enzyme under multiple environmental pressure. Protease of the present invention is better than many present advantages of using enzyme because its stability under multiple condition provides.

In fact, the protease that is in the suds is exposed to multiple washing condition, comprises the temperature of different washing agent preparations, washings volume, washings and the length of wash time. In addition, the washing agent preparation for different geographic regions has its different related component concentration at washings. For example, European washing agent generally contains the washing agent component of the 4500-5000ppm that has an appointment in washings, and Japanese washing agent generally contains the washing agent component of the 667ppm that has an appointment in washings. In North America (the particularly U.S.), washing agent generally contains the washing agent component of the 975ppm that has an appointment in washings.

Low washing agent concentration system comprises washing agent, wherein exists in washings lower than the about washing agent component of 800ppm. Japan's washing agent is considered to low washing agent concentration system usually, because there is the approximately washing agent component of 667ppm usually in they in washings.

Middle washing agent concentration system comprises washing agent, wherein exists approximately 800ppm to the about washing agent component of 2000ppm in washings. The North America washing agent is considered to middle washing agent concentration system usually, because there is the approximately washing agent component of 975ppm usually in they in washings. There is the approximately washing agent component of 1500ppm usually in Brazil in washings.

High washing agent concentration system comprises washing agent, wherein exists in washings higher than the about washing agent component of 2000ppm. The Europe washing agent is considered to high washing agent concentration system usually, because they contain the washing agent component of the 4500-5000ppm that has an appointment usually in washings.

The Latin America washing agent is generally high bubble phosphoric acid buider washing agent, and the washing agent scope that use in Latin America can fall into the high washing agent concentration of neutralization, because they contain the washing agent component of 1500ppm to 6000ppm in washings. Generally there is the approximately washing agent component of 1500ppm in Brazil in washings. Yet other high bubble phosphoric acid buider washing agent areas (being not limited only to other Latin American countries) may have to be contained up to the about high washing agent concentration system of 6000ppm washing agent component in washings.

In view of above content, obviously, washing agent composition concentration in typical case washing solution is worldwide from lower than about 800ppm washing agent composition (" low washing agent concentration area ", the approximately 667ppm of Japan for example) to about 800ppm to about 2000ppm (" middle washing agent concentration area ", the approximately 1500ppm of the approximately 975ppm of the U.S. and Brazil for example) to higher than about 2000ppm (" high washing agent concentration area ", such as the approximately 4500ppm in Europe to the about approximately 6000ppm in 5000ppm and high bubble phosphoric acid buider area), not waiting.

The concentration of typical case's washing solution is rule of thumb determined. For example, in the U.S., typical washing machine holds the approximately washing solution of 64.4L volume. Therefore, in order to obtain to wash in solution the approximately washing agent concentration of 975ppm, must be to approximately adding approximately 62.79g washing agent composition in 64.4L washing solution. This amount be the consumer use that the measuring cup that provides with washing agent weighs add typical amount in washings.

In another embodiment, different wash temperatures is used in different regions. The wash temperature of Japan is generally lower than Europe. For example, the washings temperature of North America and Japan can be 10 to 30 ℃ (for example approximately 20 ℃), and the temperature of European washings is generally 30 to 60 ℃ (for example approximately 40 ℃).

Again for example, different areas generally has the different water hardness. The Ca that the water hardness is generally mixed by per gallon²⁺/Mg ²⁺Grain (grain) number describe. Hardness is calcium (Ca in water²⁺) and magnesium (Mg²⁺) amount tolerance. Most water of the U.S. are hard, but hardness is different. Medium hardness water (60-120 ppm) to hard water (121-181ppm) has the hardness mineral matter (in Table 13-1) of 60 to 181ppm (ppm is converted to grain/USgallon: the ppm number equals grain/gallon divided by 17.1).

The water hardness in Europe is generally higher than 10.5 (for example 10.5-20.0) grain/gallon mixed C a²⁺/Mg ²⁺(the about Ca that mixes of 15 grains/gallon for example²⁺/Mg ²⁺). The water hardness of North America is generally higher than the water hardness of Japan, but lower than the water hardness in Europe. For example, the North America hardness of water can be 3 to 10 grains, 3 to 8 grains or about 6 grains. The water hardness of Japan, generally lower than the water hardness of North America, is usually less than 4, for example the Ca of 4 grains/gallon mixing²⁺/Mg ²⁺。

Therefore, in some embodiments, the invention provides at the lower protease that shows beyond thought scourability of at least one group of washing condition (for example coolant-temperature gage, the water hardness and/or washing agent concentration). In some embodiments, protease of the present invention is suitable with subtilopeptidase A aspect scourability. In some embodiments, the scourability of enhancing is compared in protease demonstration of the present invention with subtilopeptidase A. Therefore, in some preferred embodiments of the present invention, the protease that this paper provides shows oxidation stability, the heat endurance of enhancing and/or the chelating agent stability of enhancing that strengthens.

In some preferred embodiments, the invention provides homologue and the variant of ASP protease and this protease. In particularly preferred embodiments, described ASP variant comprises the multiple replacement in wild type ASP protease sequence. These protease can be used for expecting removing any application based on the spot of protein from textile or fabric.

In some embodiments, cleaning composition of the present invention is prepared into hand washing and machine washing laundry washing agent composition, comprise laundry additive combination thing, and the fabric softener composition that adds while being applicable to composition, the rinsing of pretreatment spot fabric, the composition that is used for the crust cleaning operation of general family and the operation of washing the dishes. Those skilled in the art are familiar with can be used as the different formulations of cleaning composition. In preferred embodiments, the protease of the present invention performance (that is, with other protease, comparing) that comprises quite in the washing agent composition or strengthen. In some embodiments, clean-up performance is by comparing protease of the present invention and subtilopeptidase A to assess with standard method in the multiple cleaning of utilizing enzyme sensitiveness spot (as ovum, grass, blood, breast etc.) is measured. In fact, those skilled in the art are familiar with using AAS and other analytical methods to assess the washing agent performance under the standard wash cycling condition.

Can be used for mensuration of the present invention and include but are not limited to those described in WO 99/34011 and U.S. Patent number 6,605,458 (consulting as embodiment 3). At U.S. Patent number 6,605, in 458, use the washing agent dosage of 3.0g/l in the splash bar glass beaker at the 150ml of pH 10.5,15 minutes, 15 ℃ of wash times, 6 ° of dH of the water hardness, 10nM enzyme concentration in embodiment 3, use in 50ml 5 fabric sheets (Φ=2.5cm), from EMPA 117 test materials of Center for Test Materials Holland. Use Macbeth ColorEye 7000 photometers to measure the reflectance " R " of test material at the 460nm place. Additive method provides in the embodiment of this paper. Therefore, these methods also can be used for the present invention.

Add protease of the present invention not produce any special purposes restriction in routine cleaning composition. In other words, any temperature and the pH that are applicable to this washing agent also are applicable to composition of the present invention, if this pH within scope disclosed herein and this temperature lower than described protease denaturation temperature. In addition, protease of the present invention can be used for not comprising in the cleaning composition of washing agent (be equally independent or with buider and stabilizing agent combination).

While being used for cleaning composition or washing agent, oxidation stability is the another consideration factor. Therefore, in some applications, according to multi-purpose needs, it is that strengthen, that reduce or suitable that this stability is compared with subtilopeptidase A. In some preferred embodiments, the oxidation stability of expectation enhancing. Some protease of the present invention are specially adapted to these application.

While being used for cleaning composition or washing agent, heat endurance is the another consideration factor. Therefore, in some applications, according to multi-purpose needs, it is that strengthen, that reduce or suitable that this stability is compared with subtilopeptidase A. In some preferred embodiments, the heat endurance of expectation enhancing. Some protease of the present invention are specially adapted to these application.

While being used for cleaning composition or washing agent, chelating agent stability is the another consideration factor. Therefore, in some applications, according to multi-purpose needs, it is that strengthen, that reduce or suitable that this stability is compared with subtilopeptidase A. In some preferred embodiments, expectation strengthens chelating agent stability. Some protease of the present invention are specially adapted to these application.

In some embodiments of the present invention, provide and compare the native protein enzyme that shows the enzymatic activity that changes under different pH from subtilopeptidase A. The pH activity profile is the curve of pH to enzymatic activity, can be as described embodiments and/or means known in the art build. In some embodiments, expectation obtains to have the native protein enzyme (that is, having more highly active protease in the pH scope suitable with subtilopeptidase A) of wide range. In other embodiments, this enzyme under any pH all without the activity that significantly improves, or the natural homologue (that is, have the activity of comparing enhancing with subtilopeptidase A under given pH but show in other cases more SA protease) with more sharp-pointed spectrum. Therefore, in a plurality of embodiments, protease of the present invention has different pH optimum and/or scope. The present invention is not intended to any specific pH or the pH scope of being subject to.

In some embodiments of the present invention, described cleaning composition comprises protease of the present invention, its level is 69B4 and/or other protease of the present invention of 0.00001% to 10% of composition weight, and surplus (for example 99.999% of composition weight to 90.0%), it comprises the cleaning additional materials. In other aspects of the present invention, cleaning composition of the present invention comprises 69B4 or other protease of composition weight 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% level and comprises the cleaning composition surplus that cleans additional materials (for example 99.9999% to 90.0%, 99.999% to 98%, 99.995% to 99.5%, by weight).

In some embodiments, preferably clean composition and also comprise one or more other enzymes or enzyme derivatives except protease preparation of the present invention, it provides clean-up performance and/or fabric care benefit. These enzymes include but are not limited to other protease, lipase, cutinase, amylase, cellulase, peroxidase, oxidizing ferment (for example laccase) and/or mannase.

Any other protease that are adapted at using in alkaline solution all can be used in composition of the present invention. Suitable protease comprises the protease in animal, plant or microorganism source. In particularly preferred embodiments, use the microprotein enzyme. In some embodiments, comprise the mutant of modifying with chemistry or mode of inheritance. In some embodiments, described protease is serine protease, preferred alkaline microprotein enzyme or trypsase sample protease. The example of alkali protease comprises subtilopeptidase A, particularly from those (for example subtilopeptidase A, lentus, amyloliquefaciens, subtilopeptidase A Carlsberg, subtilopeptidase A 309, subtilopeptidase As 147 and 168) of bacillus. Other examples comprise the protease mutant of describing in following patent: U.S. Patent number RE 34,606,5,955, and 340,5,700,676,6,312,936 and 6,482,628, abovely all incorporate this paper into as a reference. Other protease examples include but are not limited to the h protease of describing in trypsase (for example pig or Niu Laiyuan) and WO 89/06270. Preferred commercially available protease comprises with trade name

MAXACAL ^TM、MAXAPEM ^TM、

With

The protease that OXP (Genencor) sells; With trade name

DURAZYM ^TM、

With

(Novozymes) protease of selling; And with trade name BLAP^TMThe protease that (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf, Germany) sells. The multiple protein enzyme is described in WO95/23221, WO 92/21760 and U.S. Patent number 5,801,039,5,340,735,5,500,364,5,855,625. Other BPN ' variants (herein also referred to as " BPN '-var 1 " and " BPN ' variant 1 ") are described in US RE 34,606. Other GG36 variants (herein also referred to as " GG36-var 1 " and " GG36 variant 1 ") are described in US 5,955,340 and 5,700,676. Other GG36 variants are described in United States Patent (USP) 6,312, and 936 and 6,482,628. In one aspect of the invention, cleaning composition of the present invention comprises other protease of composition weight 0.00001% to 10% level and the cleaning additional materials of composition weight 99.999% to 90.0% level. In other embodiments of the present invention, cleaning composition of the present invention also comprise composition weight 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to the protease of 0.5%69B4 protease (or its homologue or variant) level and comprise the cleaning composition surplus that cleans additional materials (for example 99.9999% to 90.0%, 99.999% to 98%, 99.995% to 99.5%, by weight).

in addition, any lipase that is applicable in alkaline solution all can be used for the present invention. suitable lipase includes but are not limited to those of bacterium or fungi source. within the mutant of chemistry or genetic modification is also included within the present invention. the example of useful lipase comprises Humicola lanuginosa lipase (consulting as EP 258068 and EP 305216), Rhizomucor miehei (Rhizomucor miehei) lipase (consulting as EP 238023), Candida (Candida) lipase, South Pole Candida (C.antarctica) lipase (for example South Pole lipase from candida sp A or B for example, consult as EP 214761), pseudomonad (Pseudomonas) lipase, for example Pseudomonas alcaligenes (P. alcaligenes) and pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) lipase (consulting as EP 218272), Pseudomonas cepacia (P.cepacia) lipase (consulting as EP 331376), pseudomonas stanieri (P.stutzeri) lipase (is consulted as GB 1, 372, 034), Pseudomonas fluorescens (P. fluorescens) lipase, bacillus lipase is (such as bacillus subtilis lipase [Dartois etc., Biochem.Biophys.Acta 1131:253-260[1993]), bacillus stearothermophilus lipase [consulting as JP 64/744992], and bacillus brevis lipase [consulting as WO 91/16422]).

multiple clone's lipase all can be used for embodiments more of the present invention in addition, include but are not limited to penicillium cammenberti (Penicillium camembertii) lipase and (consult Yamaguchi etc., Gene 103:61-67[1991]), geotrichum candidum (Geotricum candidum) lipase (is consulted Schimada etc., J.Biochem., 106:383-388[1989]), and multiple rhizopus (Rhizopus) lipase (is consulted Hass etc. such as De Shi head mold (R.delemar) lipase, Gene 109:117-113[1991]), snow-white head mold (R.niveus) lipase (Kugimiya etc., Biosci. Biotech.Biochem.56:716-719[1992]) and Rhizopus oryzae (R.oryzae) lipase.

The lipolytic enzyme of other types (as cutinase) also can be used for embodiments more of the present invention, includes but are not limited to from the cutinase (consulting WO 88/09367) of pseudomonas mendocina (Pseudomonas mendocina) or from the cutinase (consulting WO 90/09446) of fusarium solanae (Fusarium solani pisi).

Other suitable lipase comprise commercially available lipase, for example M1 LIPASE^TM、LUMA FAST ^TMAnd LIPOMAX^TM(Genencor)； With

ULTRA (Novozymes); And LIPASE P^TM ″Amano″(Amano Pharmaceutical Co.Ltd.，Japan)。

In some embodiments of the present invention, cleaning composition of the present invention also comprise composition weight other lipase levels of 0.00001% to 10% lipase and by the surplus of the cleaning additional materials of composition weight meter. In other aspects of the present invention, cleaning composition of the present invention also comprises the lipase by composition weight meter 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% lipase level.

Any amylase (α and/or β) that is applicable in alkaline solution all can be used for embodiments more of the present invention. Suitable amylase includes but are not limited to those of bacterium or fungi source. Mutant through chemistry or genetic modification is also included within some embodiments. Can be used for amylase of the present invention and include but are not limited to the AMS (consulting as GB 1,296,839) that derives from bacillus licheniformis. Can be used for commercially available amylase of the present invention includes but are not limited to

And BAN^TM(Novozymes) and

WithP (Genencor International). In some embodiments of the present invention, cleaning composition of the present invention comprises by the amylase of composition weight meter 0.00001% to 10% other amylase levels and the surplus that cleans additional materials by composition weight meter. In other aspects of the present invention, cleaning composition of the present invention also comprises the amylase by composition weight meter 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% amylase level.

Any cellulase that is applicable to alkaline solution all can be used for embodiment of the present invention. Suitable cellulase includes but are not limited to those of bacterium or fungi source. Mutant through chemistry or genetic modification is also included within some embodiments. Suitable cellulase includes but are not limited to special detritus enzyme (Humicola insolens) cellulase (consulting as U.S. Patent number 4,435,307). Specially suitable cellulase is the cellulase (consulting as EP 0495257) with color care benefit.

Can be used for commercially available cellulase of the present invention includes but are not limited to

(Novozymes) and KAC-500 (B)^TM(Kao Corporation). In some embodiments, cellulase mixes as part or the fragment of ripe wild type or variant cellulase, has wherein lacked the N end and has divided (consulting as U.S. Patent number 5,874,276).

In some embodiments, cleaning composition of the present invention can also comprise by the cellulase of other cellulase levels of composition weight meter 0.00001% to 10% and by the surplus of the cleaning additional materials of composition weight meter. In other aspects of the present invention, cleaning composition of the present invention also comprises the cellulase by composition weight meter 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% cellulase level.

Any mannase that is applicable to alkaline solution all can be used for the present invention. Suitable mannase includes but are not limited to those of bacterium or fungi source. Mutant through chemistry or genetic modification is also included within some embodiments. Known multiple mannase can be used for the present invention's (consult as U.S. Patent number 6,566,114, U.S. Patent No. 6,602,842 and U.S. Patent number 6,440,991, all incorporate this paper into as a reference).

In some embodiments, cleaning composition of the present invention can also comprise by the mannase of composition weight meter 0.00001% to 10% other mannosan enzyme levels and by the surplus of the cleaning additional materials of composition weight meter. In other aspects of the present invention, cleaning composition of the present invention also comprises the mannase by composition weight meter 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% mannosan enzyme level.

In some embodiments, peroxidase and hydrogen peroxide or its source (for example percarbonate, perborate or persulfate) are used in combination. In alternative embodiment, oxidizing ferment and oxygen are used in combination. This enzyme of two types all is used for " solution bleaching " (namely, prevent that fabric textile dyestuff when washing is washed in liquid together is transferred to other fabrics from the fabric of dyeing), preferably use (consulting as WO 94/12621 and WO 95/01426) together with synergist. Suitable peroxidase/oxidizing ferment includes but are not limited to those of plant, bacterium or fungi source. Mutant through chemistry or genetic modification is also included within some embodiments.

In some embodiments, cleaning composition of the present invention can also comprise by the peroxidase of other peroxidase of composition weight meter 0.00001% to 10% and/or oxidizing ferment level and/or oxidizing ferment and by the surplus of the cleaning additional materials of composition weight meter. Of the present invention aspect other in, cleaning composition of the present invention also comprises peroxidase and/or the oxidizing ferment by composition weight meter 0.0001% to 10%, 0.001% to 5%, 0.001% to 2%, 0.005% to 0.5% peroxidase and/or oxidizing ferment level.

The mixture of above-mentioned enzyme is also included within herein, particularly the mixture of 69B4 enzyme, one or more other protease, at least a amylase, at least a lipase, at least a mannase and/or at least a cellulase. In fact, the mixture of multiple these enzymes is all considered for the present invention.

The varying level of protease and one or more other enzymes all can reach 10% independently, and the surplus of cleaning composition is the cleaning additional materials. Easily determine after the expectation form that specifically is chosen in the composition of considering surface, article or fabric to be cleaned and be used for clean conditions when using (for example by cleaning washing agent, using) of cleaning additional materials.

the example of suitable cleaning additional materials includes but are not limited to surfactant, buider, bleaching agent, the bleaching activator, bleaching catalyst, other enzymes, enzyme stabilization system, chelating reagent, fluorescent whitening agent, soil release polymer, the dyestuff transfer agent, dispersant, defoamer, dyestuff, aromatic, colouring agent, fill salt, hydrotropic agent, light activating agent, fluorescer, fabric regulator, hydrolyzable surfactant, anticorrisive agent, antioxidant, antishrinking agent, anti wrinkling agent, bactericide, fungicide, color spot (color speckles), silver care agent (silvercare), anti-dark and gloomy dose and/or corrosion inhibitor, the basicity source, solubilizer, carrier, processing aid, pigment and pH controlling agent (are consulted as

U.S. Patent number

6, 610, 642, 6, 605, 458, 5, 705, 464, 5, 710, 115, 5, 698, 504, 5, 695, 679, 5, 686, 014 and 5, 646, 101, all incorporate this paper into as a reference). hereinafter detailed example the embodiment of concrete cleaning composition material.

, if the cleaning additional materials is incompatible with of the present invention protease of cleaning in composition, use suitable method will clean that additional materials separates (that is, not contacting with each other) with protease until this two kinds of components while being suitable for making up. Such separating method comprises any known method in this area (such as soft capsule, encapsulation, tablet, physics separation etc.).

Preferably, provide one or more protease to be included in this paper of effective dose to can be used for and clean multiple needs and remove in the composition on surface of albumen spot. Such cleaning composition comprises the cleaning composition for these application, and it is used for cleaning crust, fabric and bowl dish. In fact, in some embodiments, the invention provides fabric cleaning composition, and in other embodiments, the invention provides non-woven cleaning composition. It should be noted that the present invention also provides the cleaning that is suitable for personal hygiene composition, comprise MC (comprise denfifrice, toothpaste, collutory etc., and cleaning of teeth composition), skin and hair cleaning composition. The present invention is intended to comprise the washing agent composition of any form (being liquid, particle, bar shaped, semisolid, gel, emulsion, tablet, capsule etc.).

For example, some cleaning compositions that can use protease of the present invention have hereinafter been described in more detail. Clean in the embodiment that composition is prepared as the composition that is applicable to the washing of drum type washing machine method in the present invention, composition of the present invention preferably contains at least a surfactant and at least a buider compound, and one or more cleaning additional materials, it is preferably selected from organic polyhydroxyl compound, bleaching agent, other enzymes, defoamer, dispersant, lime soap dispersant, soil suspending agent and anti redeposition agent and corrosion inhibiter. In some embodiments, the laundry composition also contains softening agent (that is, as other cleaning additional materials).

Composition of the present invention can also be used for the washing agent additive product of solid or liquid form. These additive products are intended to supplement and/or strengthen the performance of conventional washing agent composition, and can add in any stage of washing process.

In the embodiment that is prepared as for the composition of the manual method that washes the dishes, composition of the present invention preferably contains at least a surfactant, and preferably contain at least a other cleaning additional materials, it is selected from organic polyhydroxyl compound, foam improver, II family metal ion, solvent, helps aqueous solvent and other enzymes.

In some embodiments, the density of the laundry washing agent composition of this paper is 400 to 1200g/ liters, and in other embodiments, is 500 to 950g/l compositions while measuring under 20 ℃.

In some embodiments, multiple cleaning composition (, as U.S. Patent number 6,605, providing in 458) can use together with protease of the present invention. Therefore, in some embodiments, the composition that comprises at least a protease of the present invention is fine and close graininess fabric cleaning composition, and in other embodiments, described composition is the graininess fabric cleaning composition that can be used for washing colored fabric, and in other embodiments, described composition is for providing softening graininess fabric cleaning composition in washability, in other embodiments, described composition is high power capacity liquid fabric cleaning composition.

In some embodiments, the composition that comprises at least a protease of the present invention is fabric cleaning composition, as U.S. Patent number 6,610, and 642 and 6,376, described in 450. In addition, protease of the present invention is used under Europe or Japanese washing condition in useful especially particulate laundry detergent composition (consulting as United States Patent (USP) 6,610,642).

In alternative embodiment, the invention provides the crust cleaning composition that comprises protease that at least a this paper provides. Therefore, in some embodiments, the composition that comprises at least a protease of the present invention is crust cleaning composition, as U.S. Patent number 6,610, and 642, U.S. Patent number 6,376,450 and U.S. Patent number 6,376, those described in 450.

In other embodiments, the invention provides the composition that washes the dishes that comprises protease that at least a this paper provides. Therefore, in some embodiments, the composition that comprises protease that at least a this paper provides is crust cleaning composition, as U.S. Patent number 6,610, and 642 and U.S. Patent number 6,376, those described in 450.

In other embodiments, the invention provides the composition that washes the dishes that comprises protease that at least a this paper provides. Therefore, in some embodiments, the composition that comprises protease that at least a this paper provides comprises oral care composition, as U.S. Patent number 6,610, and 642 and U.S. Patent number 6,376, those described in 450.

The formula that comprises in above-mentioned U.S. Patent number 6,376,450,6,605,458,6,605,458 and 6,610,642 and to compound and the cleaning additional materials description all incorporate clearly this paper into as a reference. Other embodiment are disclosed in embodiment hereinafter.

In addition, the invention provides composition and method for the production of food or animal feed, it is characterized in that protease of the present invention is mixed with food or animal feed. In some embodiments, above-mentioned protease adds as dryed product in first being processed, and in other embodiments, and it is in first being processed or add as liquid afterwards. In some embodiments of using dry powder, enzyme is diluted on dry support (grain that for example grinds) as liquid. Protease of the present invention can be used as the component of animal feed and/or additive, as U.S. Patent number 5,612, and 055, U.S. Patent number 5,314,692 and U.S. Patent number 5,147, those described in 642, they all incorporate this paper into as a reference.

Enzyme feed addictive of the present invention is suitable for preparing with several different methods. For example, in some embodiments, it mixes to produce the enzyme mixture by the different enzymes that will have suitable activity simply and prepares. In some embodiments, this enzyme mixture directly mixes with feed, and in other embodiments, and it is impregnated on carrier material (as the wheat that grinds, corn or soy meal) based on cereal. The present invention also comprises these carriers through dipping, because they can be used as the enzyme feed addictive.

In some alternative embodiment, will flood simultaneously or successively the enzyme with suitable activity based on the carrier (wheat that for example grinds or corn) of cereal. For example, in some embodiments,, with the wheat carrier that the grinds zytase of at first spraying, be then protease, and optional 1,4 beta-glucanase. The present invention also comprises these carriers through dipping, because they can be used as the enzyme feed addictive. In preferred embodiments, these carriers through dipping comprise at least a protease of the present invention.

In some embodiments, feed addictive of the present invention directly mixes with animal feed, and in alternative embodiment, other feed addictives of itself and one or more mix, and described other feed addictives are vitamin feed additive, mineral additive for feed and/or amino acid feed additive for example. The gained feed addictive that then will comprise several dissimilar components mixes with feed with suitable amount.

In some preferred embodiments, the feed addictive of the present invention that comprises based on the carrier of cereal generally mixes with following amount: 0.01-50g/kg feed, more preferably 0.1-10g/kg, most preferably from about 1g/kg.

In alternative embodiment, enzyme feed addictive of the present invention relates to the restructuring microorganism that builds the enzyme of expecting with the relative quantity generation of expectation. In some embodiments, this by raising encode at least a protease of the present invention gene copy number and/or by using the suitable strong promoter that effectively is connected with the polynucleotides of this protease of encoding to realize. In other embodiments, described restructuring microorganism bacterial strain has lacked some enzymatic activity (such as cellulase, endoglucanase etc.) as required.

In other embodiments, enzyme feed addictive provided by the invention also comprises other enzymes, includes but are not limited at least a zytase, AMS, glucoamylase, pectase, mannonase alpha-galactosidase, phytase and/or lipase. In some embodiments, the enzyme that will have a required activity and zytase and protease mix before on they being impregnated into based on the carrier of cereal, perhaps with these enzymes simultaneously or be impregnated into successively on such carrier based on cereal. Then more described carrier is mixed to prepare final feed with the feed based on cereal. In alternative embodiment, described enzyme feed addictive is prepared as the solution of each enzymatic activity, then with the feed material that is pre-formed as piller or pastel, mixes.

In other embodiments, described enzyme feed addictive is included in animal diet followed, and this is by realizing in the drinking water that it is mixed the second (for example different) feed or animal. Therefore, enzyme mixture provided by the invention must not mix the feed itself based on cereal, but such mixing forms particularly preferred embodiment of the present invention. The ratio of the active unit of zytase/g feed addictive and proteinase activity unit/g feed addictive is preferably 1: 0.001-1,000, more preferably 1: 0.01-100, most preferably 1: 0.1-10. As described above, enzyme mixture provided by the invention preferably is used as feed addictive in the preparation of the feed based on cereal.

In some embodiments, described feed based on cereal comprises at least 25% weight or the wheat of preferred at least 35% weight or the combination of corn or these two kinds of cereal. Described feed also comprises protease (for example at least a protease of the present invention), and its amount makes this feed comprise the proteinase activity/kg of 100-100000 unit.

Feed based on cereal provided by the invention can be used as the feed for multiple non-human animal, and described animal comprises poultry (such as turkey, goose, duck, chicken etc.), domestic animal (such as pig, sheep, ox, goat etc.) and pet (such as horse, dog, cat, rabbit, mouse etc.). Described feed is specially adapted to poultry and pig, particularly broiler chicken.

The present invention also is provided for processing the composition of textile, and it comprises at least a protease of the present invention. In some embodiments, at least a protease of the present invention is the component (consulting as U.S.'s second edition patent No. 216,034, EP 134,267, U.S. Patent number 4,533,359 and EP 344,259) that is applicable to process in the composition of silk or wool.

In addition, protease of the present invention can be used for expecting the multiple application of separation of phosphorus from phytate. Therefore, the present invention also provides to produce and has the wool that improves characteristic or the method for animal wool material. In some preferred embodiments, these methods comprise the following steps: by being selected from technique pretreatment wool, wool fiber or the animal wool material of plasma-treating technology and Delhey technique; And pretreated wool or animal wool material are processed with the proteolytic enzyme (for example at least a protease of the present invention) that effectively improves characteristic quantity. In some embodiments, described proteolytic enzyme is processed and was carried out before plasma treatment, and in other embodiments, it carries out after plasma treatment. In other embodiments, it carries out as independent step, and in other embodiments, and scouring or the dyeing combination of itself and described wool or animal wool material are carried out. In other embodiments, there are at least a surfactant and/or at least a softening agent in the enzyme treatment step, and in other embodiments, surfactant and/or softening agent are mixing in step separately, in this step, wool or animal wool material are softened processing.

In some embodiments, composition of the present invention can be used in the method for anti-shrinkage wool fiber (consulting the 4-327274 as JP). In some embodiments, described composition is used for, by following steps, wool fiber is carried out the method that anti-shrinkage is processed: fiber is carried out Low Temperature Plasma Treating, use anti-shrinkage resin (for example block urethane resin, polyamine epichlorohydrin resin, glyoxalic acid resin, ethylidene-urea resin or acrylic resin) to process, process to obtain bating effect with the proteolytic enzyme of weight reduction more afterwards thereafter. In some embodiments, described plasma treatment step is K cryogenic treatment, is preferably Corona discharge Treatment or glow discharge and processes.

In some embodiments, described Low Temperature Plasma Treating is carried out with gas, is preferably selected from the gas of air, oxygen, nitrogen, ammonia, helium or argon. Usually using air, can be favourable but use the described gas of any gas.

Preferably, described Low Temperature Plasma Treating was carried out approximately 2 seconds to approximately 300 seconds to the pressure of 5 holders in about 0.1 holder, and preferably approximately 5 seconds to approximately 100 seconds, more preferably from about 5 seconds to approximately 30 seconds.

As described above, the present invention can use (consulting as DE-A-4332692) with the Combination of Methods such as Delhey technique. In this technique, under existing, soluble tungstate salt processes wool in aqueous hydrogen peroxide solution, randomly process in the solution of synthetic polymer or dispersion thereafter, to improve the nonshrink suede characteristic of wool. In the method, catalyst (preferred Na in 2-60% (weight/weight), preferred 8-20% (weight/weight))₂WO ₄) exist lower and under the nonionic wetting agent exists, process wool in the aqueous solution of hydrogen peroxide (0.1-35% (weight/weight), preferred 2-10% (weight/weight)). Preferably, this processing is carried out under pH 8-11 and room temperature. Processing time is depended on the concentration of hydrogen peroxide and catalyst, but preferred 2 minutes or shorter. This oxidation is used the water rinse wool after processing. , in order to remove remaining hydrogen peroxide and randomly to carry out extra bleaching, further process wool in the acid solution of reducing agent (for example sulphite, phosphite).

In some embodiments, described enzyme treatment step carried out approximately 1 minute to approximately 120 minutes. This step is preferably at approximately 20 ℃ to approximately 60 ℃, and more preferably from about 30 ℃ are extremely approximately carried out at the temperature of 50 ℃. Perhaps, wool is immersed in or loads in the aqueous solution of enzyme, then carry out the decatize typing under conventional temperature and pressure, generally processed 30 seconds to approximately 3 minutes. In some preferred embodiments, described proteolytic enzyme is processed and is carried out in the acidity that can comprise buffer or neutrality or alkaline medium.

In alternative embodiment, described enzyme treatment step is at one or more conventional anionics, nonionic (Dobanol for example; Henkel AG) or under the cationic surface active agent existence carry out. The example of useful nonionic surface active agent is Dobanol (from Henkel AG). In other embodiments, described wool or animal wool material are carried out ultrasonic processing, it carried out or with it, carried out simultaneously before proteolytic enzyme is processed. In some preferred embodiments, described ultrasonic processing was approximately being carried out approximately 5 minutes at the temperature of 50 ℃. In some preferred embodiments, the amount of the proteolytic enzyme that uses in the enzyme treatment step is for about 0.2 weight/% by weight to about 10 weight/% by weight, and it is based on the weight of wool or animal wool material. In some embodiments,, in order to reduce number of steps, in the dyeing of wool or animal wool material and/or in cleaning, process by carrying out enzyme in protease being added dyeing, rinsing and/or cleaning liquid simply. In some embodiments, enzyme is processed and is carried out after plasma treatment, but in other embodiments, these two treatment steps carry out with reverse order.

The conventional normally cationic softening agent of softening agent that is used for wool, be organic cation type softening agent or silica-based product, but also can use anion or nonionic softening agent. The example of useful softening agent includes but are not limited to the softening agent of polyethylene and silicone softening agent (that is, dimethyl silicone polymer (silicone oil)), H-polysiloxanes, silicone elastomer, the dimethyl silicone polymer of amino functional, the silicone elastomer of amino functional and dimethyl silicone polymer and the organic cation type softening agent (for example alkyl quaternary ammonium salts derivative) of epoxide function.

In other embodiments, the invention provides the composition for the treatment of animal hides, it comprises at least a protease of the present invention. In some embodiments, protease of the present invention can be used for processing the composition of animal hides, described in WO 03/00865 (Insect Biotech Co., Taejeon-Si, Korea). In other embodiments, the invention provides for rawhide and/or skin being processed into the method for leather, it comprises that with protease of the present invention, described rawhide or skin being carried out enzyme processes (consulting as WO 96/11285). In other embodiments, the invention provides for animal skins or rawhide being processed into the composition of leather, it comprises at least a protease of the present invention.

Rawhide and skin in tannery generally with through pickling or dry rawhide or skin form obtains. The method that rawhide or skin is processed into leather comprises some different procedure of processings, comprises immersion, depilation and softening step. These steps have formed wet processing, and carry out in beamhouse. Utilizing the enzyme of protease of the present invention to process can any time application during related method in leather processing. Yet generally (namely soak, depilation and/or softening process in) uses protease in wet process. Therefore, in some preferred embodiments, described enzyme is processed in wet process segment process and is carried out the enzyme processing with at least a protease of the present invention.

In some embodiments, soaking technology of the present invention (for example pH is pH 6.0-11) under conventional soaking conditions carries out. In some preferred embodiments, pH is 7.0-10.0. In alternative embodiment, temperature is 20-30 ℃, and in other embodiments, is preferably 24-28 ℃. In other embodiments, the reaction time is 2-24 hour, and is preferably 4-16 hour. In other embodiments, provide as required surfactant and/or anticorrisive agent.

The second stage of softening step is usually to add softening agent itself to start. In some embodiments, enzyme is processed and is carried out in softening process. In some preferred embodiments, enzyme is processed and is carried out in softening process after the stage at deliming. In some embodiments, softening technique of the present invention uses normal condition to carry out (for example pH is pH 6.0-9.0). In some preferred embodiments, the pH scope is 6.0-8.5. In other embodiments, temperature is 20 to 30 ℃, and in preferred embodiments, temperature is 25 to 28 ℃. In some embodiments, the reaction time is 20 to 90 minutes, and in other embodiments, is 40 to 80 minutes. Technique for the production of leather is well known to those skilled in the art (consults as WO 94/069429, WO 90/1121189, U.S. Patent number 3,840,433, EP 505920, GB 2233665 and U.S. Patent number 3,986,926, all incorporate this paper into as a reference).

In other embodiments, the invention provides the softening agent that comprises at least a protease of the present invention. Softening agent is reagent or the enzyme-containing formulation that comprises for the chemical active ingredient of beamhouse technique (especially for the softening technique of producing leather). In some embodiments, the invention provides the softening agent that comprises protease and suitable excipient. In some embodiments, reagent includes but are not limited to chemicals known in the art and that use, for example diluent, softening agent, deliming agent and carrier. In some embodiments, prepare the softening agent (consulting as GB-A2250289, WO 96/11285 and EP 0784703) that comprises at least a protease of the present invention as known in the art.

In some embodiments, softening agent of the present invention contains 0.00005 to 0.01g active protease/g softening agent, and in other embodiments, described softening agent contains 0.0002 to 0.004g active protease/g softening agent.

Therefore, protease of the present invention can be used in multiple application and situation.

Experiment

The present invention is described in greater detail in following embodiment, and they are not intended to limit the scope of the invention by any way.Accompanying drawing should be thought the intact part of specification sheets of the present invention and description.All reference that this paper quotes are all incorporated this paper into as a reference especially.Provide following examples to be illustrative rather than definitive thereof the present invention.

To use following abbreviation: PI (protease inhibitors) in experiment disclosure hereinafter; Ppm (1,000,000/); M (mole/liter); MM (mM/l); μ M (micromole/liter); NM (nanomole/liter); Mol (mole); Mmol (mM); μ mol (micromole); Nmol (nanomole); Gm (gram); Mg (milligram); μ g (microgram); Pg (pik); L (liter); Ml and mL (microlitre); μ l and μ L (microlitre); Cm (centimetre); Mm (millimeter); μ m (micron); Nm (nanometer); U (unit); V (volt); MW (molecular weight); Sec (second); Min (minute); H and hr (hour); ℃ (degree centigrade); QS (effective dose); ND (not carrying out); NA (inapplicable); Rpm (revolutions per minute); H₂O (water); dH₂O (deionized water); HCl (hydrochloric acid); Aa (amino acid); Bp (base-pair); Kb (kilobase to); KD (kilodalton); CDNA (copy or complementary DNA); DNA (DNA); SsDNA (single stranded DNA); DsDNA (double-stranded DNA); DNTP (deoxyribonucleotide triphosphoric acid); RNA (ribonucleic acid); MgCl₂(magnesium chloride); NaCl (sodium chloride); W/v (weight/volume); V/v (volume/volume); G (gravity); OD (optical density); Dulbecco phosphate buffer (DPBS); SOC (2% bacto-tryptone, 0.5% bacterium yeast extract, 10mM NaCl, 2.5mM KCl); Terrific nutrient solution (TB; 12g/l bacto-tryptone, 24g/l glycerine, 2.31g/l KH₂PO ₄With 12.54g/l K₂HPO ₄)； OD ₂₈₀(optical density at 280nm place); OD₆₀₀(optical density at 600nm place); A₄₀₅(absorbance at 405nm place); Vmax (the highest initial speed of enzymic catalytic reaction); PAGE (polyacrylamide gel electrophoresis); PBS (PBS [150mM NaCl, 10mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25%

20); PEG (polyethylene glycol); PCR (PCR); RT-PCR (reverse transcription PCR); SDS (lauryl sodium sulfate); Tris (three (methylol) aminomethane); HEPES (the N-[2-ethoxy] piperazine-N-[2-ethyl sulfonic acid]); HBS (HEPES BS); Tris-HCl (three [methylol] aminomethane-hydrochloride); Tricine (N-[three (methylol)-methyl]-glycine); CHES (2-(the N-cyclohexyl is amino) ethyl sulfonic acid); TAPS (3-{[three (methylol)-methyl]-amino }-propane sulfonic acid); CAPS (3-(cyclohexyl is amino)-propane sulfonic acid; DMSO (methyl-sulfoxide); DTT (Isosorbide-5-Nitrae-two sulfo-s-DL-threitol); SA (sinapic acid (s, 5-dimethoxy-4 '-hydroxycinnamic acid); TCA (trichloroacetic acid); Glut and GSH (reduced glutathione); GSSG (oxidized form of glutathione); TCEP (three [2-carboxyethyl] phosphine); Ci (Curie); MCi (millicurie); μ Ci (micromicrocurie); HPLC (high pressure liquid chromatography (HPLC)); RP-HPLC (reverse phase HPLC chromatography); TLC (thin-layer chromatography); MALDI-TOF (mastrix-assisted laser desorption ionization time of flight Ji Zhifuzhude of Matrix-assisted); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (thermus aquaticus (Thermus aquaticus) archaeal dna polymerase); Klenow (large (Klenow) fragment of DNA polymerase i); EGTA (ethylene glycol-two (beta-amino ether) N, N, N ', N '-tetraacethyl); EDTA (ethylenediamine tetra-acetic acid); Bla (beta-lactamase or ampicillin resistance gene); HDL (high density liquid); MJ Research (MJ Research, Reno, NV); Baseclear (Baseclear BV, Inc., Leiden, Holland); PerSeptive (PerSeptive Biosystems, Framingham, MA); ThermoFinnigan (ThermoFinnigan, San Jose, CA); Argo (Argo BioAnalytica, Morris Plains, NJ); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, NY); EMPA Testmaterialien AG (EMPA Testmaterialien AG, St.Gallen-Winkeln, Switzerland); Warwick Equest (Warwick Equest Limited, Durham, UK); Minolta (Konica Minolta, Inc., Japan); United States Testing (United States Testing Co, Inc, Hoboken, NJ); Spectrum (Spectrum Laboratories, Dominguez Rancho, CA); Molecular Structure (Molecular Structure Corp., Woodlands, TX); Accelrys (Accelrys, Inc., San Diego, CA); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, NJ); CFT (Center for Test Materials, Vlaardingen, the Netherlands); Procter﹠Gamble (Procter﹠Gamble, Inc., Cincinnati, OH); GE Healthcare (GE Healthcare, Chalfont St.Giles, United Kingdom); DNA2.0 (DNA2.0, Menlo Park, CA); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, DE); Corning (Corning Life Sciences, Corning, NY); (NEN (NEN Life Science Products, Boston, MA); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, TX); ATCC (American Type Culture Collection, Rockville, MD); Gibco/BRL (Gibco/BRL, Grand Island, NY); Sigma (Sigma Chemical Co., St.Louis, MO); Pharmacia (Pharmacia Biotech, Piscataway, NJ); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, CA); BD Biosciences and/or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, CA); Operon Technologies (Operon Technologies, Inc., Alameda, CA); MWG Biotech (MWG Biotech, High Point, NC); Oligos Etc (Oligos Etc.Inc, Wilsonville, OR); Bachem (Bachem Bioscience, Inc., King of Prussia, PA); Difco (Difco Laboratories, Detroit, MI); Mediatech (Mediatech, Herndon, VA; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); Oxoid (Oxoid Inc., Ogdensburg, NY); Worthington (Worthington Biochemical Corp., Freehold, NJ); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, MD); Millipore (Millipore, Billerica, MA); Bio-Rad (Bio-Rad, Hercules, CA); Invitrogen (Invitrogen Corp., San Diego, CA); NEB (New England Biolabs, Beverly, MA); Sigma (Sigma Chemical Co., St. Louis, MO); Pierce (Pierce Biotechnology, Rockford, IL); Takara (Takara Bio Inc.Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, NJ); Qiagen (Qiagen, Inc., Valencia, CA); Biodesign (Biodesign Intl., Saco, Maine); Aptagen (Aptagen, Inc., Herndon, VA); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, NC); Molecular Devices (Molecular Devices, Corp., Sunnyvale, CA); R﹠D Systems (R﹠D Systems, Minneapolis, MN); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Marsh (Marsh Biosciences, Rochester, NY); Geneart (Geneart GmbH, Regensburg, Germany); Bio-Tek (Bio-Tek Instruments, Winooski, VT); (Biacore (Biacore, Inc., Piscataway, NJ); PeproTech (PeproTech, Rocky Hill, NJ); SynPep (SynPep, Dublin, CA); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, NJ); Waters (Waters, Inc., Milford, MA); Matrix Science (Matrix Science, Boston, MA); Dionex (Dionex, Corp., Sunnyvale, CA); Monsanto (Monsanto Co., St.Louis, MO); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, NJ); Huntsman (Huntsman Petrochemical Corp., Salt Lake City, UT); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, the Netherlands); Dow Corning (Dow Corning Corp., Midland, MI); And Microsoft (Microsoft, Inc., Redmond, WA).

The wild-type serine protease that uses in following examples is described in detail in US04/39006 and US04/39066, and they are all complete incorporates this paper into as a reference.In addition, the embodiment 2 of U.S. Patent Application Serial 10/576,331 provides the details that produces 69B4 proteolytic enzyme from Gram-positive alkaliphile 69B4.Described in the application's full text, this priority application integral body is incorporated this paper into as a reference.

Embodiment 1

Assay method

In following examples, used multiple assay method, for example protein determination, based on the test of using and based on the test of stability.Hereinafter provide and in corresponding embodiment, indicate for being easy to read, below being determined at.Hereinafter any deviation of the scheme that any experiment provides of carrying out in the performance history of the present invention is all indicated in an embodiment.

Some washing composition that use in following examples have following composition.In composition I and II, surplus (to 100%) is spices/dyestuff and/or water.The pH of these compositions for about 5 to about 7, is about 7.5 to about 8.5 for composition I I for composition I.In composition I II, surplus (to 100%) comprises water and/or a small amount of spices, dyestuff, whitening agent/SRPI/ Xylo-Mucine/optical white/MgSO ₄/ PVPVI/ defoamer/high molecular weight PEGs/clay.

A. the TCA that is used for protein content determination in 96 hole microtiter plates measures

Use the filtration culture supernatants of shaking with 230 RPM under comfortable 33 ℃ and under the humidity ventilation condition, cultivating 4 days to begin this mensuration.96 new hole flat undersides are used for this mensuration.At first, the 0.25N HCl with 100 μ L/ holes places the hole.Then, Xiang Kongzhong adds the filtered nutrient solution of 50 μ L.Then measure the scattering of light/absorbancy (in plate reader, using 5 seconds mixed modes) at 405nm place, so that " blank " reading to be provided.

For testing, 15% (w/v) TCA in 100 μ L/ holes is placed flat board and at room temperature hatched 5 to 30 minutes.Then measure the scattering of light/absorbancy (in plate reader, using 5 seconds mixed modes) at 405nm place.

Calculate by from the TCA test reading, deducting blank (promptly not having TCA's).In case of necessity, can measure calibration TCA reading by AAPF, to produce typical curve with clone with known transition factor.Yet TCA result is linear with regard to 50 to 500ppm protein concn, therefore can be directly to the enzyme performance mapping, to be used to select the purpose of high-performance variant.

B. the suc-AAPF-pNA that carries out proteolytic enzyme in 96 hole microtiter plates measures

In this mensuration system, the reagent solution of use is as follows:

1.100mM Tris/HCl, pH 8.6, contain 0.005%

-80 (Tris damping fluids)

2.100mM the Tris damping fluid, pH 8.6, contain 10mM CaCl ₂With 0.005%

-80 (Tris damping fluids)

3.DMSO in 160mM suc-AAPF-pNA (suc-AAPF-pNA stock solution) (Sigma:S-7388)

In order to prepare the suc-AAPF-pNA working fluid, add 1ml AAPF stoste in the 100ml Tris damping fluid and at least 10 seconds of thorough mixing.

Following mensuration: in each hole, add the protein enzyme solution of 10 μ l dilution, thereafter (rapidly) adding 190 μ l 1mg/ml AAPF working fluids.Solution was mixed for 5 seconds, change in the absorbancy of in the MTP reader, reading 410nm under 25 ℃.Protease activity is expressed as AU (activity=Δ ODmin ^-1.ml ^-1).

C. the Keratin sulfate hydrolysis is measured

In this mensuration system, the chemical and the reagent solution of use are as follows:

Keratin sulfate ICN 902111

Washing composition is dissolved in (pH=8.2) in the 1000ml water with the 1.6g washing composition

The 0.6ml CaCl that also adds 10000gpg ₂/ MgCl ₂And 1190mg

HEPES provides the hardness and the buffering intensity that are respectively 6gpg and 5mM.

Is 8.2 with NaOH with pH regulator.

Picryl sulfonic acid (Picrylsulfonic acid, TNBS)

Sigma P-2297 (5% aqueous solution)

Reagent A is with 45.4g Na ₂B ₄O ₇.10 H ₂The 4NNaOH of O (Merck 6308) and 15ml is molten together to 1000ml final volume (heating in case of necessity)

Reagent B is with 35.2g NaH ₂PO ₄.1H ₂O (Merck 6346) and 0.6g Na ₂SO ₃(Merck 6657) are molten together to the 1000ml final volume.

Method:

Before hatching, Keratin sulfate is filtered with 100 μ m filters at every turn on a small quantity.Then, at room temperature the Keratin sulfate with 10g＜100 μ m stirred 20 minutes in detergent solution at least, and the pH routine is adjusted to 8.2.At last, under the room temperature with centrifugal 20 minutes of suspension (Sorvall, the GSA rotor, 13,000rpm).Then repeat this operation.At last, with moistening throw out resuspended suspension that stirs to the 200ml cumulative volume and in moving the liquid process in washing composition always.Before hatching, with Biohit hyperchannel pipettor and 1200 μ l suction nozzles with microtiter plate (microtiter plate, MTP) each hole 200 μ l substrates (distribute 6 200 μ l, rapidly divide to be equipped with as much as possible and avoids Keratin sulfate in suction nozzle, to precipitate) of packing in.Then, in the MTP that contains substrate, add the filtering culture of 10 μ l.Use the tape upper flat plate, place incubator and cultivate down 3 hours (Innova 4330[NewBrunswick]) at 20 ℃ with 350rpm.After the cultivation, with flat board at centrifugal 3 minutes of 3000rpm (iSigma 6K 15 whizzers).Before first flat board is taken out from incubator about 15 minutes, mix 1ml TNBS solution by every 50ml reagent A and prepare RNBS reagent.

Each hole 60 μ l TNBS reagent of packing in MTP.From the flat board of cultivating, 10 μ l are transferred among the MTP that contains the TNBS reagent A.With the tape upper flat plate and in desk-top shaking table (BMG Thermostar), shaking 20 minutes under room temperature and the 500rpm.At last, Xiang Kongzhong adds 200 μ l reagent B, mixes 1 minute on shaking table, measures the absorbancy of 405nm with the MTP reader.

Calculate the Keratin sulfate hydrolytic activity:

Proofread and correct the gained absorbancy with blank value (substrate of no enzyme).The gained absorbancy provides the tolerance of hydrolytic activity.To each sample (variant) calculation of performance indicators.The performance of variant (actual value) and standard enzyme (theoretical value) under the described performance index comparison same protein concentration.In addition, can use the calculation of parameter theoretical value of bright wrong your (Langmuir) equation of standard enzyme.Performance index greater than 1 (PI) (PI＞1) are represented better variant (comparing with standard substance (as wild-type)), represent the performance variant identical with standard substance and PI is 1 (PI=1), and on behalf of performance, the PI less than 1 (PI＜1) be worse than the variant of standard substance.

Therefore, PI represented the person of winning and not too expectation be used for variant under some situation.

D. the little sample (Microswatch) that is used for the test proteins enzyme performance is measured

All washing composition that use during these are measured all do not contain enzyme.

Detergent formulations:

1. cold water liquid washing agent (U.S.'s condition):

Milli-Q water is adjusted to the water hardness (Ca/Mg=3/1) of 6gpg, adds the 1.60g/l washing composition, with detergent solution vigorous stirring at least 15 minutes.Then, add 5mM Hepes damping fluid and with pH regulator to 8.2.Before being used for this mensuration, detergent use 0.22 μ m filter (for example Nalgenetop bottle filter) is filtered.

2. low pH liquid washing agent (U.S.'s condition):

Milli-Q is adjusted to the water hardness (Ca/Mg=3/1) of 6gpg, adds the 1.60g/l washing composition

-LVJ-1 or

2005) or the 1.50g/l washing composition ( -SNOW), with detergent solution vigorous agitation at least 15 minutes.Using 1N NaOH solution is 6.0 with pH regulator.Before being used for this mensuration, detergent use 0.22 μ m filter (for example Nalgene top bottle filter) is filtered.

Little sample:

Order little sample of 1/4 inch ring-type diameter, supply by CFT Vlaardingen.Using hereinafter described, fixing means carries out pre-treatment to little sample.Single little samples vertical is placed each holes of 96 hole microtiter plates, to expose whole surf zone (that is, be not be tiled in hole at the bottom of).

" 3K " sample fixes:

This specific sample is fixing at room temperature to carry out.Yet, add 30%H ₂O ₂Amount be 10 times of super fixing (superfixed) sample fixation method (promptly carrying out) of using under the European condition with 60 ℃.It is visible that foam forms (foaming), therefore must use bigger beaker.At first, 8 liters of distilled water are placed the beaker of 10L, add 80ml 30% hydrogen peroxide.With spoon with water and hydrogen peroxide thorough mixing.Then, then add in the solution 40 EMPA, 116 sample launch into sector, evenly fixing to guarantee.Sample was stirred (use spoon) 30 minutes in solution, stirred continuously in preceding 5 minutes, intermittently stirred in all the other 25 minutes.Discard solution and, use about 6 liters of distilled water at every turn sample rinsing 6 times.Place on the paper handkerchief sample dry.Use 1/4 inch circular die on the extruder (expulsion press) that air-dry sample is punched.Single little samples vertical is placed each holes of 96 hole microtiter plates, to expose whole surf zone (that is, be not be tiled in hole at the bottom of).

The enzyme sample:

With each geographic proper concn tested enzyme sample, and be diluted to 10mM NaCl, 0.005%

In-80 solution.

Testing method:

Incubator is arranged to desired temperatures: following 20 ℃ of cold fluid concrete conditions in the establishment of a specific crime, or following 30 ℃ of low pH liquid condition.Pre-treatment and the sample that cuts are in advance placed the hole of 96 hole MTP, as above-mentioned.In case of necessity, with the enzyme sample at 10mM NaCl, 0.005% Be diluted to 20 * expectation concentration in-80.Detergent solution as above-mentioned preparation expectation.Then, 190 μ l detergent solutions are added in each hole of MTP.In this mixture of each Kong Zhongxiang, add 10 μ l enzyme solution (so that the cumulative volume in 200 μ l/ holes to be provided).With dull and stereotyped sealer sealing MTP and hatched 60 minutes, stir with 350rpm.After hatching under proper condition, from each hole, take out 100 μ l solution and place new MTP.In the MTP reader, the new MTP that contains 100 μ l solution/holes is carried out reading at the 405nm place.Also comprise blank and contained little sample and washing composition but do not contain the contrast of enzyme.Stock solution is with 15,000gpg (3: 1 (1.92M Ca of Ca/Mg ²⁺=282.3g/L CaCl ₂.2H ₂O; 0.64MMg ²⁺=30.1g/L MgCl ₂.6H ₂O) concentration is used.

Detergent composition and incubation conditions in the little sample determination of table 1-1

Washing composition cold water liquid	Reference enzyme ASP	Washing composition 1.5 or 1.6g/l washing composition	Water hardness 6gpg Ca/Mg:3/1	Enzyme amount-[ppm] 0.3-4	20 ° of temperature (℃ .)	Sample 3K
Washing composition cold water liquid	Reference enzyme ASP	Washing composition 1.5 or 1.6g/l washing composition	Water hardness 6gpg Ca/Mg:3/1	Enzyme amount-[ppm] 0.3-4	20 ° of temperature (℃ .)	Sample 3K	Low pH liquid washing agent	ASP	1.6g/l washing composition	6gpg Ca/Mg：3/1	-0.5-4	30°	3K

Calculate the BMI performance

The absorbance of gained is proofreaied and correct with blank value (obtaining after hatching little sample under the no enzyme situation).The absorbancy of gained is the tolerance of hydrolytic activity.To each sample (variant) calculation of performance indicators.The performance of variant (actual value) and standard enzyme (theoretical value) under the described performance index comparison same protein concentration.In addition, can use the calculation of parameter theoretical value of bright wrong your equation of standard enzyme.Performance index greater than 1 (PI) (PI＞1) are represented better variant (comparing with standard substance (as wild-type)), represent the performance variant identical with standard substance and PI is 1 (PI=1), and on behalf of performance, the PI less than 1 (PI＜1) be worse than the variant of standard substance.

D. the dimethyl casein hydrolysis is measured (96 hole)

Dimethyl casein (DMC): Sigma C-9801

-80： Sigma P-8074

PIPES buffer reagent (anacidity): Sigma P-1851; 15.1g is dissolved in about 960ml water; With

4N NaOH is adjusted to 7.0 with pH, adds 1ml 5%

-80 and volume complemented to 1000ml.PIPES

With

-80 final concentration be respectively 50mM and

0.005％。

Picryl sulfonic acid (TNBS): Sigma P-2297 (5% aqueous solution)

Reagent A is with 45.4g Na ₂B ₄O ₇.10H ₂O (Merck 6308) and 15ml

4N NaOH molten together to the 1000ml final volume (in case of necessity

Heating)

Reagent B is with 35.2g NaH ₂PO ₄.1H ₂O (Merck 6346) and 0.6g

Na ₂SO ₃(Merck 6657) are molten together to the 1000ml end-body

Long-pending.

Method:

In order to prepare substrate, 4g DMC is dissolved in the 400ml PIPES damping fluid.Filtered culture supernatants is diluted with the PIPES damping fluid, and the final concentration that contrasts in the culture plate is 20ppm.Then with in the 200 μ l substrates in the supernatant liquor adding MTP hole of every kind of dilution of 10 μ l.With MTP plate on the tape, shook several seconds, and place 37 ℃ baking oven 2 hours, and do not stir.

First flat board was being taken out from baking oven preceding approximately 15 minutes, mixing 1ml TNBS solution by every 50ml reagent A and prepare RNBS reagent.Each hole 60 μ l TNBS reagent of packing in MTP.The flat board of hatching was shaken several seconds, afterwards 10 μ l are transferred among the MTP that contains the TNBS reagent A.With the tape upper flat plate and in desk-top shaking table (BMG Thermostar), shaking 20 minutes under room temperature and the 500rpm.At last, Xiang Kongzhong adds 200 μ l reagent B, mixes 1 minute on shaking table, measures the absorbancy of 405nm with the MTP reader.

Calculate dimethyl casein hydrolysis activity:

The absorbance of gained is proofreaied and correct with blank value (substrate of no enzyme).The absorbancy of gained is the tolerance of hydrolytic activity.By come (arbitrarily) specific activity of calculation sample divided by the protein concn that records with absorbancy.

E. thermal stability determination

This mensuration is based on before the heating buffered culture supernatants and dimethyl casein hydrolysis afterwards.Use and identical chemical and reagent solution described in the dimethyl casein hydrolysis mensuration.

Method:

Filtered culture supernatants is diluted to 20ppm (based on the concentration that contrasts in the grown cultures plate) in the PIPES damping fluid.At first, the enzyme sample of every kind of dilution is got 10 μ l be determined at the initial activity of dimethyl casein in measuring, and as following the processing.Then, the supernatant liquor of every kind of dilution of 50 μ l is placed the emptying aperture of MTP.The MTP plate was hatched 90 minutes with 400rpm with 60 ℃ in iEMS incubator/shaking table HT (Thermo Labsystems).With flat board cooled on ice 5 minutes.Then, this solution adding of 10 μ l is contained among the new MTP in 200 μ l dimethyl casein substrate/holes the final activity after hatching with mensuration.With this MTP on the tape, shook several seconds and place 37 ℃ baking oven 2 hours and do not stir.Use with the DMC hydrolysis and measure identical detection method.

Calculate thermostability:

The residual activity of sample is expressed as the ratio of final absorbancy and initial absorbancy (all blank being proofreaied and correct).

F.LAS stability is measured

Measure LAS stability after in the presence of 0.06%LAS (Sodium dodecylbenzene sulfonate), hatching the test proteins enzyme, use AAPF to measure and measure residual activity.

Reagent:

Pelopon A (=LAS): Sigma D-2525

-80：Sigma P-8074

TRIS damping fluid (anacidity): Sigma T-1378); 6.35g is dissolved in about 960ml water, uses 4N HCl pH regulator to 8.2.The TRIS final concentration is 52.5mM.

LAS stock solution: preparation 10.5%LAS solution (=10.5g/100ml MQ) in MQ water

TRIS damping fluid-100mM/pH 8.6 (100mM Tris/0.005%Tween80)

The TRIS-Ca damping fluid, pH 8.6 (100mM Tris/10mM CaCl ₂/ 0.005%Tween80) hardware:

Flat MTP:Costar (#9017)

Biomek FX

The ASYS multichannel pipettor

Spectramax MTP reader

IEMS incubator/shaking table

Innova 4330 incubators/shaking table

The Biohit multichannel pipettor

BMG Thermostar shaking table

Method:

Preparation 0.063%LAS solution in 52.5mM Tris pH of buffer 8.2.By 1ml100mg/ml AAPF stock solution (among the DMSO) being added 100ml (100mM) TRIS damping fluid, prepare the AAPF working fluid among the pH 8.6.In order to dilute supernatant liquor,, add supernatant liquor aliquots containig and thorough mixing with the dilution buffer liquid of packing in the flat flat board.Thinning ratio depends on the concentration (AAPF activity) of ASP contrast in the culture plate.Desirable protein matter concentration is 80ppm.

The supernatant liquor of 10 μ l dilution is added in 190 μ l 0.063%LAS damping fluid/holes.With MTP on the tape, shook several seconds and placed the incubator (Innova 4230) 60 minutes of 25 ℃ or 35 ℃, shake with 200rpm.After hatching 10 minutes,, 10 μ l mixtures in each hole measure initial activity (t=10 minute) among the new MTP that contains 190 μ l AAPF working fluids by being transferred to.With these solution thorough mixing, and use MTP reader (in 5 minutes He under 25 ℃, reading 20 readings) to measure the AAPF activity.

Measure final active (t=60 minute) by after hatching 60 minutes, again 10 μ l solution being taken out from hatch flat board.As above-mentioned mensuration AAPF activity.Following calculating:

The % residual activity is [t-60 value] * 100/[t-10 value].

Embodiment 2

In subtilis, produce ASP proteolytic enzyme

In the present embodiment, in subtilis, experimentize as described to produce 69B4 proteolytic enzyme (being also referred to as " ASP ", " Asp " and " ASP proteolytic enzyme " and " Asp proteolytic enzyme " herein).In the present embodiment, described plasmid pHPLT-ASP-C1-2 (see figure 1) has been transformed in the subtilis.Transform (consult as WO 02/14490, incorporate this paper into as a reference) as known in the art.In order to optimize the expression of ASP in subtilis, produce the synthetic dna sequence dna by DNA2.0, and be used for these expression experiments.Dna sequence dna (synthetic ASP dna sequence dna) encoding wild type ASP precursor protein with the codon selection that is applicable to bacillus species provided below:

ATGACACCACGAACTGTCACAAGAGCTCTGGCTGTGGCAACAGCAGCTGCTACACTCTTG

GCTGGGGGTATGGCAGCACAAGCT AACGAACCGGCTCCTCCAGGATCTGCATCAGCCCC

TCCACGATTAGCTGAAAAACTTGACCCTGACTTACTTGAAGCAATGGAACGCGATCTGGG

GTTAGATGCAGAGGAAGCAGCTGCAACGTTAGCTTTTCAGCATGACGCAGCTGAAACGG

GAGAGGCTCTTGCTGAGGAACTCGACGAAGATTTCGCGGGCACGTGGGTTGAAGATGAT

GTGCTGTATGTTGCAACCACTGATGAAGATGCTGTTGAAGAAGTCGAAGGCGAAGGAGC

AACTGCTGTGACTGTTGAGCATTCTCTTGCTGATTTAGAGGCGTGGAAGACGGTTTTGGA

TGCTGCGCTGGAGGGTCATGATGATGTGCCTACGTGGTACGTCGACGTGCCTACGAATTC

GGTAGTCGTTGCTGTAAAGGCAGGAGCGCAGGATGTAGCTGCAGGACTTGTGGAAGGCG

CTGATGTGCCATCAGATGCGGTCACTTTTGTAGAAACGGACGAAACGCCTAGAACGATG

TTCGACGTAATTGGAGGCAACGCATATACTATTGGCGGCCGGTCTAGATGTTCTAT

CGGATTCGCAGTAAACGGTGGCTTCATTACTGCCGGTCACTGCGGAAGAACAGGAG

CCACTACTGCCAATCCGACTGGCACATTTGCAGGTAGCTCGTTTCCGGGAAATGAT

TATGCATTCGTCCGAACAGGGGCAGGAGTAAATTTGCTTGCCCAAGTCAATAACTA

CTCGGGCGGCAGAGTCCAAGTAGCAGGACATACGGCCGCACCAGTTGGATCTGCTG

TATGCCGCTCAGGTAGCACTACAGGTTGGCATTGCGGAACTATCACGGCGCTGAAT

TCGTCTGTCACGTATCCAGAGGGAACAGTCCGAGGACTTATCCGCACGACGGTTTG

TGCCGAACCAGGTGATAGCGGAGGTAGCCTTTTAGCGGGAAATCAAGCCCAAGGTG

TCACGTCAGGTGGTTCTGGAAATTGTCGGACGGGGGGAACAACATTCTTTCAACCA

GTCAACCCGATTTTGCAGGCTTACGGCCTGAGAATGATTACGACTGACTCTGGAAG

TTCCCCT GCTCCAGCACCTACATCATGTACAGGCTACGCAAGAACGTTCACAGGAACCC

TCGCAGCAGGAAGAGCAGCAGCTCAACCGAACGGTAGCTATGTTCAGGTCAACCGGAGC

GGTACACATTCCGTCTGTCTCAATGGACCTAGCGGTGCGGACTTTGATTTGTATGTGCAG

CGATGGAATGGCAGTAGCTGGGTAACCGTCGCTCAATCGACATCGCCGGGAAGCAATGA

AACCATTACGTACCGCGGAAATGCTGGATATTATCGCTACGTGGTTAACGCTGCGTCAGG

ATCAGGAGCTTACACAATGGGACTCACCCTCCCCTGA(SEQ ID NO：10)

In above sequence, the DNA of runic indication encoding mature proteolytic enzyme, standard letter indication leader sequence, underscore indication N end and C hold former sequence.

Express synthetic ASP gene

Make up the Asp expression cassette in pXX-KpnI (see figure 2) carrier, its rear clone advances pHPLT carrier (see figure 3), to be used for expressing ASP subtilis.PXX-KpnI is based on the carrier of pUC, contains the copy aprE promotor that drives aprE promotor (subtilis), the cat gene of expressing and be used for increasing subtilis copy number.The Bla gene allows selective growth in intestinal bacteria.The KpnI that introduces in the ribosome bind site of aprE promoter region downstream can clone the Asp expression cassette with the HindIII site is feasible in pXX-KpnI.

PHPLT-EBS2c2 (derivative of a kind of pHPLT) (Solingen etc., Extremophiles5:333-341[2001]) contain the thermally-stabilised amylase LAT promotor (PLAT) of Bacillus licheniformis, be thereafter XbaI and the HpaI restriction site that is used to clone the ASP expression construct.

The Asp expression cassette is cloned in the pXX-KpnI carrier of the DNA that contains coding heterozygosis signal peptide, described heterozygosis signal peptide is constructed by 5 subtilopeptidase A prE N end signal peptide ammino acids and 25 Asp C end signal peptide ammino acids and forms (MRSKKRTVTRALAVATAAATLLAGGMAAQA; SEQ ID NO:11).Transform in the intestinal bacteria (ElectromaxDH10B, Invitrogen, catalog number (Cat.No.) 12033-015) being cloned into Asp expression cassette in the pXX-KpnI carrier.The primer that uses and clone's strategy provide in table 2-1.Thereafter, this expression cassette of clone from these carriers, and introduce in the pHPLT expression vector, be used for transforming subtilis (Δ aprE, Δ nprE, oppA, Δ spoIIE, degUHy32, Δ amyE::(xylR, pxylA-comK) bacterial strain.ASP expression cassette clone's primer and clone's strategy provide in table 2-2 among the pHPLT.As incorporate the conversion of carrying out described in this paper WO 02/14490 as a reference in subtilis into.

Primer derives from MWG and Invitrogen.Scheme according to Invitrogen is used for pcr amplification (0.2 μ M primer, 25 to 30 circulations) with InvitrogenPlatinum Taq high-fidelity DNA polymerase (catalog number (Cat.No.) 11304-029).The scheme that the ligase enzyme reaction of ASP expression cassette and host's carrier uses Invitrogen T4 dna ligase (catalog number (Cat.No.) 15224-025) to utilize Invitrogen to recommend to be used for sticking terminal general clone is finished.

In bacillus subtilis strain (Δ aprE, Δ nprE, oppA, Δ spoIIE, degUHy32, Δ amyE::(xylR, pxylA-comK.) middle research asp expression of gene.With plasmid pHPLT-ASP-C1-2 (seeing Table 2-2 and Fig. 1) transform subtilis (Δ aprE, Δ nprE, oppA, Δ spoIIE, degUHy32, Δ amyE::(xylR, pxylA-comK) in.Transform (consult as WO 02/14490, it incorporates this paper into as a reference) as known in the art.

Containing 25ml Synthetic Maxatase substratum (SMM) (with 0.97g/lCaCl ₂.6H ₂O replaces 0.5g/l CaCl ₂(consult U.S. Patent number 5,324,653, incorporate this paper into as a reference), and contain the 20mg/L Xin Meisu) shake subtilis (the Δ aprE that will contain the pHPLT-ASP-C1-2 carrier in the bottle, Δ nprE, oppA, Δ spoIIE, deg UHy32, Δ amyE::(xylR, pxylA-comK) transformant is carried out selective growth.This growth causes producing the secretor type ASP proteolytic enzyme with proteolytic activity.Use NuPage Novex 10%Bis-Tris gel (Invitrogen, catalog number (Cat.No.) NP0301BOX) to carry out gel analysis.In order to prepare the sample that is used to analyze, the 4 * LDS sample buffer (Invitrogen, catalog number (Cat.No.) NP0007) and the 1%PMSF (20mg/ml) of the supernatant liquor of 2 times of volumes and the 1M HCl of 1 times of volume, 1 times of volume mixed, thereafter 70 ℃ of heating 10 minutes.Then, every kind of sample of 25 μ L and 10 μ L SeeBlue plus, 2 pre-dsred protein standard substance (Invitrogen, catalog number (Cat.No.) LC5925) are gone up sample together in gel.The result shows that clearly the described asp clone of present embodiment strategy makes producing bacillus subtilis liveliness proof Asp.

In addition, the sample of the same fermented liquid of following mensuration: get the supernatant liquor of 10 μ l dilution and join that (concentration 1mg/ml is at 0.1M Tris/0.005% in the 190 μ l AAPF substrate solutions

Among the pH 8.6).Monitor the rising speed (25 ℃) of the 410nm absorbancy that causes owing to liberate p-nitroaniline, it provides the tolerance of the ASP concentration that produces.These results show that subtilis pHPLT-ASP-C1-2 transformant causes producing measurable ASP proteolytic enzyme.

Embodiment 3

Make up combination mutant

The structure in ASP variant multiple mutation library has been described in the present embodiment.Contain as the ASP variant of the skeleton in multiple mutation library and to replace R014I-A064K-T086K-T116E-R123F.This variant is cloned in the pHPLT carrier.Use

Multidigit point directed mutagenesis (QCMS) test kit (Stratagene) makes up this library.5 ' the phosphorylation primer that is used to produce this library is shown in table 3-1.The primer of noticing HPLC, PAGE or any other type purifying provides much better result aspect the wrong primer mixing the total length primer and significantly reduce to contain.Yet, in these experiments, do not use the primer of purifying.

The pUC18-ASP preparation

Use PstI and HindIII restriction site, will contain the ASP variant of replacing R014I-A064K-T086K-T116E-R123F and advance pUC18 carrier (Invitrogen, catalog number (Cat.No.) 15363013 from the pHPLT carrier cloning; See Fig. 1).PUC18-ASP plasmid (see figure 2) electroporation advanced electroreception attitude Bacillus coli cells (Invitrogen, catalog number (Cat.No.) C4040-52, One thereafter,

TOP10 Electrocomp ^TME.coli, dam+) in, and selective growth on the agar plate that contains the 100mg/L penbritin produces the Bacillus coli cells that has the pUC18-ASP plasmid.This method is used to guarantee to carry out the ASP DNA that methylates of the required GATC site of QCMS scheme, because plasmid pHPLT-ASP-C1-2 does not grow in intestinal bacteria.

Prepare DNA in a small amount from the Bacillus coli cells preparation that contains the pUC18-ASP plasmid.Particularly, with bacterial strain overnight incubation in containing 10mL 2 * TY substratum of 100ppm penbritin, thereafter cell centrifugation is got off.Use Qiagen spin miniprep DNA test kit (catalog number (Cat.No.) 27106), prepare plasmid DNA by the step of pointing out in the Qiagen miniprep test kit handbook.Prepare DNA in a small amount with the 50uL Qiagen buffering EB wash-out that provides in this test kit.

The structure in multiple mutation library

Following selection is used for the site of multiple mutation library construction.For each required in final molecule characteristic, for be defined as productive site sudden change is evaluated as " on " or D score.At least, it is productive that each desired characteristic is tackled in site to be made up, and is productive to most important characteristic at least.Have the maximum probability that improves or keep all key properties in order to ensure the library, use following standard.

For each characteristic, set up the cutoff of Δ Δ G.For example, for the method for carrying out under 25 ℃, on behalf of these sudden changes, Δ Δ G value＜0.06Kcal be not less than 90% of parent's protein-active, and on behalf of these, Δ Δ G value＞1.13Kcal sport 125% of parent's protein-active.

The primer concentration that in reaction, uses, all as structure multiple mutation library as described in the Stratagene QCMS test kit.Particularly, the methylated purifying pUC18-ASP of 1 μ L plasmid (about 70ng) is mixed with 15 μ L sterile distilled waters, 1.5 μ L dNTP, 2.5 μ L, 10 * damping fluid, 1 μ L enzyme mixture and 1.0 μ L mutant primer mixtures (amounting to the 100pmol primer).Use that every kind 10 μ L prepares primer mixture (100pmol/ μ L) in 18 kinds of mutant primers; Add every kind of primer that 50ng is used for the library as the recommendation of Stratagene, take turns the less sudden change of generation in the mutagenesis several before.Therefore, in current mutagenesis round, revise this scheme and amount to the 100pmol primer in each reactant, to comprise.Cycling condition be 95 ℃ 1 minute, be thereafter 95 ℃ 1 minute, 55 ℃ 1 minute, 65 ℃ 30 circulations of 12 minutes, use thin-walled 0.2mL PCR pipe in MJ ResearchPTC-200 thermal cycler, to carry out.By 37 ℃ of overnight incubation, use 1 μ L DpnI digestion reaction product from the QCMS test kit.Add 0.5 μ L DpnI again, and reactant was hatched 1 hour.

Thereafter, with library DNA (the strand pUC18-ASP product of mutagenesis) electroporation to electroreception attitude Bacillus coli cells (Invitrogen, catalog number (Cat.No.) C4040-52, One

TOP10Electrocomp ^TME.coli dam+) and selective growth on the agar plate that contains the 100mg/L penbritin, produces ASP multiple mutation library in intestinal bacteria.Results bacterium colony (tens of) to thousands of, and use Qiagen spin miniprep DNA test kit (catalog number (Cat.No.) 27106), prepare plasmid DNA by the listed step of Qiagen miniprep test kit handbook.The DNA for preparing in a small amount with the 50uL Qiagen damping fluid EB wash-out that provides in this test kit.

Use PstI and the HindIII DNA restriction enzyme digestion DNA of preparation in a small amount.With the ASP library fragment mixture (gel-purified of PstI * HindIII), and utilize Invitrogen to recommend to be used for general sticking terminal clone's scheme, advance in the HindIII * PstI pHPLT carrier segments of 4154 base pairs by the ligase enzyme reaction clone who uses Invitrogen T4 dna ligase (catalog number (Cat.No.) 15224-025) to carry out.In other method, produce synthetic ASP library fragment by GeneArt.These ASP library fragments are also with PstI and HindIII digestion, and purifying also reacts the clone by ligase enzyme and advances in the HindIII * PstI pHPLT carrier segments of 4154 base pairs.

For the ligation mixture is directly transformed in the bacillus cell, use TempliPhi test kit (Amersham catalog number (Cat.No.) 25-6400) amplification library DNA (clone advances the ASP library fragment mixture among the pHPLT)., 1 μ L ligation mixture is mixed from the sample buffer of TempiPhi test kit with 5 μ L for this reason, and heat 3 minutes with denatured DNA at 95 ℃.Reactant was placed cooled on ice 2 minutes, follow of short duration centrifugal., add 5 μ L reaction buffers and 0.2 μ L phi29 polysaccharase from TempliPhi test kit, reactant was hatched under 30 ℃ 4 hours in MJ Research PCR instrument thereafter.Made phi29 enzyme heat inactivation in reaction in 10 minutes by in the PCR instrument, hatching with 65 ℃.

In order the library to be transformed in the genus bacillus, with 0.1 μ L TempliPhi amplified reaction product and 500 μ L competence bacillus subtilis mycetocyte (Δ aprE, Δ nprE, oppA, Δ spoIIE, degUHy32, Δ amyE::(xylR pxylA-comK) mixes, and acutely shakes under 37 ℃ 1 hour thereafter, 100 and 500 μ L are seeded in contain 20ppm neomycinsulphate (Sigma, catalog number (Cat.No.) N-1876; Contain 732 μ g Xin Meisus in every gram) and the HI agar plate of 0.5% skimming milk on.95 clones of picking are used for order-checking from the library.

Mutagenesis is carried out very smoothly, because have only 14% clone identical with frame sequence (ASP that contains R014I-A064K-T086K-T116E-R123F), extra sudden change has taken place about 3% clone.Remaining order-checking clone (72%) is mutant, and wherein about 94% is unique mutant.The sequencing result in this library provides in table 3-3.

Embodiment 4

The preparation of the rough enzyme sample of ASP variant enzyme

By in 96 hole MTP, subtilis transformant (above with described in the United States Patent (USP) sequence number 10/576,331) being produced the Asp misfolded proteins in 68 hours cultivating under 37 ℃ in MBD substratum (based on the defined medium of MOPS).Substantially as known in the art prepare MBD substratum (consult]), just in minimum medium, got rid of NH as Neidhardt etc., J.Bacteriol., 119:736-747[1974 ₄Cl ₂, FeSO ₄And CaCl ₂, use 3mM K ₂HPO ₄And in minimum medium, replenish 60mM urea, 75g/L glucose and 1% soya peptone.Simultaneously, micro-nutrients is prepared into 100 * stock solution, it contains 400mg FeSO in 1 liter ₄.7H ₂O, 100mg MnSO ₄.H ₂O, 100mg ZnSO ₄.7H ₂O, 50mg CuCl ₂.2H ₂O, 100mgCoCl ₂.6H ₂O, 100mg NaMoO ₄.2H ₂O, 100mg Na ₂B ₄O ₇.10H ₂O, 10ml 1MCaCl ₂With 10ml 0.5M Trisodium Citrate.

In following examples, the test that multiple ASP mutant is carried out has been described.Use the above method described in the embodiment 1.In following table, " variant code " provides the amino acid (that is, " F001A " is illustrated in this specific variants, and 1 phenylalanine is replaced by L-Ala in the aminoacid sequence) of wild-type amino acid, the position in aminoacid sequence and replacement.

Embodiment 5

The casein hydrolysis activity of multiple replacement variant

In the present embodiment, the active experiment of casein hydrolysis that is used to measure multiple multiple replacement ASP variant has been described.In these experiments, the scheme of use embodiment 1 as mentioned is described.Following table provides variant (comprising the sudden change in each variant) and activity to be higher than the active casein of the variant of wild-type ASP+1 times standard deviation (〉=1.13 activity unit).

Following table provides variant (comprising the sudden change in each variant) and activity to be higher than the active casein of the variant of wild-type ASP+1 times standard deviation (〉=0.85 activity unit).

These results show that the performance of multiple multiple replacement variant in this mensuration system is better than wild-type ASP.

Embodiment 6

The Keratin sulfate hydrolytic activity of multiple replacement variant

The experiment of the Keratin sulfate hydrolytic activity that is used to measure multiple multiple replacement ASP variant has been described in the present embodiment.In these experiments, the scheme of use embodiment 1 as mentioned is described.Following table provides variant (comprising the sudden change in each variant) and activity to be higher than the Keratin sulfate hydrolytic activity of the variant of wild-type ASP+1 times standard deviation (〉=1.1 performance index).

Following table provides variant (comprising the sudden change in each variant) and activity to be higher than the Keratin sulfate activity of the variant of wild-type ASP+1 times standard deviation (＞1.2 performance index).

Embodiment 7

The thermostability of multiple replacement variant

In the present embodiment, the experiment of carrying out for the thermostability of the ASP variant of measuring multiple multiple replacement has been described.In these experiments, the scheme of use embodiment 1 as mentioned is described.Following table provides variant (comprising the sudden change in each variant) and activity to be higher than the remaining active casein of the variant of wild-type ASP+1 times standard deviation (〉=48% residual activity).

Embodiment 8

The LAS stability of multiple replacement variant

The experiment of the LAS stability that is used to measure multiple multiple replacement ASP variant has been described in the present embodiment.In these experiments, the scheme of use embodiment 1 as mentioned is described, uses 25 ℃ temperature.Following table provides variant (comprising the sudden change in each variant) and residual activity to be higher than the remaining AAPE activity of the variant of wild-type ASP+1 times standard deviation (〉=10% residual activity).

In other experiment, tested the LAS stability of variant with multiple replacement.In first group of experiment, under 25 ℃ temperature, use embodiment 1 described test condition.In following table, showed that activity is higher than the variant of wild-type+1 times standard deviation (＞20% residual activity).

In other experiments, used other test condition.In one group of experiment, use embodiment 1 described scheme to test LAS stability down in higher temperature (35 ℃).In following table, showed that activity is higher than the residual activity percentage ratio of the variant of wild-type+1 times standard deviation (＞5% residual activity).

In other embodiments, use embodiment 1 described scheme to test variant down at 35 ℃.In following table, showed contain G12D and R35E and＞result of the variant of 70% residual activity.

In other experiments, other conditions have been used.In one group of experiment, use embodiment 1 described scheme to measure the performance index of multiple multiple replacement variant, 25 ℃ of tests down.In following table, showed that residual activity is higher than the performance index result (PI) of the variant of wild-type+1 times standard deviation (＞1.1 performance index).

In other embodiments, 25 ℃ of LAS stability of using embodiment 1 described scheme to test multiple variant down.As above-mentioned, choice criteria is that residual activity is higher than WT+1 times of standard deviation (＞1.1 performance index).

Embodiment 9

The soil release performance of multiple replacement mutant

In these experiments, use embodiment 1 described scheme as mentioned.Following table provides variant, comprises that activity is higher than the sudden change in each variant of wild-type+1 times standard deviation (＞1.1 performance index [PI]).These results show that the performance of multiple multiple replacement variant in this mensuration system is better than wild-type ASP.

In other experiments of using liquid washing agent, obtained following result.As shown in the table, the variant that marks in the following table shows the activity that is higher than wild-type+1 times standard deviation (＞1.1 performance index).

In other are implemented, obtained following result.In these experiments, with variant with compare as the 0.5ppm wild-type ASP of reference.The variant that activity is higher than wild-type+1 times standard deviation (＞1.1 performance index) provides the result in the following table.

In other experiments, obtained following result.The variant that marks in the following table shows the activity that is higher than wild-type+1 times standard deviation (＞1.1 performance index).

Embodiment 10

The soil release performance of multiple replacement variant

The experiment of the soil release ability that is used to measure multiple multiple replacement ASP variant has been described in the present embodiment.In these experiments, use following scheme and material.

At first, by the calcium chloride (CaCl that at first 11.020g/L dewatered ₂.2H ₂O) MW 147.01g/mol and 5.08g/L Magnesium dichloride hexahydrate (MgCl ₂.6H ₂O) MW 203.3g/mol is dissolved in the 1000ml deionized water and prepares 10,000ppm stock solution (3/1Ca ^{+ 2}/ Mg ^{+ 2}) prepare 105ppm water (～6gpg).With 10.50ml 10, the 000ppm stock solution dilutes the solution for preparing 105ppm with the 989.50ml deionized water.

By with 11.2gm

-2005 are dissolved in 7 liters of 105ppm water and prepare in the 105ppm water

2005 detergent solutions (1.6gm/L).

Use anti-water-color paintbrush that the following sample that uses in these experiments is numbered (on dirty face): from the sample EMPA 116 (11x8cm), the EMPA117 (11x8cm) that make dirty with blood-breast-ink (BMI) of EMPA Testmaterialien AG; And the sample EMPA164 (10x7.5cm) that makes dirty from the grass of EMPA Testmaterialien AG and from the Equest grass medium of the Warwick Equest Limited sample (10x7.5cm) of making dirty.On white background, use the dirty face of Tristimulus MinoltaMeter CR-300 (Minolta) measure sample, and use equation L (L ^*a ^*B), the D65 standard illuminants is analyzed.Each sample carries out reading three times.Because careless spot is very responsive to light, therefore the sample that grass is made dirty covers and keeps in Dark Place stand-by.Each washing forms test and all carries out in duplicate.

In test, used extra ballast weight (more mechanical effect).This ballast weight comprises 2 10x10cm EMPA 221 placing 1 beaker (not Wu Zhuo cotton, EMPATestmaterialien AG).These EMPA 221 samples are not carried out label and measurement.

The test of laundry scourability uses the desk-top Tergotometer of 6 cylinders (TOM) 7243S types (United States Testing) to carry out.Temperature regulation is 15 ℃ or 60 °F.Half of TOM inboard is equipped with ice.Washing time is set to 12 minutes.Test the 1 liter of detergent solution (in 105ppm water, preparing, as indicated above) of packing in the beaker.In case solution reaches 15 ℃ or 60 °F, TOM just begins to stir with 100rpm.Then as the variant sample is joined in the mixture hereinafter pointedly.Use does not add any enzyme sample

2005 detergent solution (Procter﹠amp; Gamble) in contrast.Variant is tested under two different concns (being 0.55ppm and 2.75ppm).

In each beaker, add 6 samples (having identical spot), whenever next.In addition, add 2 EMPA 221 samples.For example:, be 3 EMPA116 and 3 EMPA117+2EMPA 221 (gross weight is about 13.25 grams) for the sample that BMI makes dirty; , be 3 EMPA 164 and 3 sample+2EMPA 221 (gross weight is about 13.22 grams) that Equest grass medium is made dirty perhaps for the grass sample of making dirty.Washing time is re-set as 12 minutes.Wash after 12 minutes, the rinsing in bucket with all EMPA 116 and EMPA 117 samples, the rinsing in another bucket of all careless samples, rinsing is 3 minutes in cold mobile tap water.Then discard EMPA 221 samples.Sample was placed centrifugal dryer 2 minutes (EMPA 116 and EMPA 117 samples are dry respectively with the sample that grass is made dirty).Thereafter the sample that grass is made dirty is air-dry in air, does not carry out illumination or flatiron, and EMPA 116 and EMPA 117 samples are then pressed dried.

After the drying, on white background, use Tristimulus Minolta Meter CR-300 with equation L (L ^*a ^*B), the dirty face of sample is assessed in the D65 standard illuminants.Use following Equation for Calculating soil release per-cent:

% soil release=(the L value before the L value-washing after the washing)/(L _{0 white cotton}L value before the-washing) * 100%

In order to obtain the dirty tolerance of removing of each variant, then deduct under no any variant existence and pass through by the soil release per-cent under existing with every kind of variant

The soil release percentage that 2005 detergent solutions are obtained recently calculates Δ soil release per-cent (Δ %SR).The results are shown in table 10.1.

In other experiments, tested the soil release ability of multiple multiple replacement ASP variant.Use test condition mentioned above, wherein carry out following modification.In these tests, by with 37.5gm

2005SNOW is dissolved in 25 liters of 105ppm water and prepares in the 105ppm water

2005SNOW detergent solution (1.5gm/L).In addition, the sample that EMPA 116BMI is made dirty is cut into the small pieces of 10 * 7.5cm.The EMPA 117 samples are replaced with EMPA 116 fixed samples (CFT), also be cut into the small pieces of 10 * 7.5cm.In these experiments, do not use the EMPA164 sample.Under the concentration of 0.55ppm, test variant.In each beaker, add 6 samples (that is, 6

EMPA

116 or 6 EMPA 116 fixed or 6 Equest), add one at every turn.In addition, add 2 EMPA 221 samples.Use does not add any enzyme sample

2005 SNOW detergent solution (Procter﹠amp; Gamble) in contrast.Each scourability test is all carried out in quadruplicate.Result's (average delta %SR, standard deviation (STD) and repeat number (n)) is shown in Fig. 6.

Table 10.1 variant/dosage	EMPA11 Δ％S.R. (n＝6)	EMPA 117 Δ％S.R (n＝6)	Equest grass Δ %S.R. (n=6)	EMPA 164 Δ％S.R. (n＝6)
Table 10.1 variant/dosage	EMPA11 Δ％S.R. (n＝6)	EMPA 117 Δ％S.R (n＝6)	Equest grass Δ %S.R. (n=6)	EMPA 164 Δ％S.R. (n＝6)	ASP/0.55ppm	5.2±1.2	4.9±2.4	-4.3±4.3	1.5±1.1
ASP/2.75ppm	9.7±1.4	10.7±2.	-1.8±5.4	3.1±1.6	ASP/0.55ppm	5.2±1.2	4.9±2.4	-4.3±4.3	1.5±1.1
ASP/2.75ppm	9.7±1.4	10.7±2.	-1.8±5.4	3.1±1.6	R123L-R127Q-R179Q/0.55ppm	6.0±1.5	8.5±2.1	1.7±3.7	2.1±1.2
R123L-R127Q-R179Q/2.75ppm	11.3±1.5	14.0±2.	1.1±5.0	5.4±1.4	R123L-R127Q-R179Q/0.55ppm	6.0±1.5	8.5±2.1	1.7±3.7	2.1±1.2
R123L-R127Q-R179Q/2.75ppm	11.3±1.5	14.0±2.	1.1±5.0	5.4±1.4	G12D-R35H/0.55ppm	7.0±1.1	7.9±1.9	-2.2±5.0	2.5±1.3
G12D-R35H/2.75ppm	10.1±1.7	14.4±1.	3.2±3.8	4.5±1.0	G12D-R35H/0.55ppm	7.0±1.1	7.9±1.9	-2.2±5.0	2.5±1.3
G12D-R35H/2.75ppm	10.1±1.7	14.4±1.	3.2±3.8	4.5±1.0	G12D-R35E/0.55ppm	8.2±1.2	10.0±2.	1.3±5.4	2.9±1.4
G12D-R35E/2.75ppm	10.6±1.4	16.6±2.	4.3±4.2	5.4±1.4	G12D-R35E/0.55ppm	8.2±1.2	10.0±2.	1.3±5.4	2.9±1.4
G12D-R35E/2.75ppm	10.6±1.4	16.6±2.	4.3±4.2	5.4±1.4	R35E-R123L-R127Q-R179Q/0.5 ppm	6.1±1.2	6.8±2.1	0.4±4.1	1.0±1.4
R35E-R123L-R127Q-R179Q/2.7 ppm	8.8±2.1	12.7±1.	4.7±4.3	1.7±1.4	R35E-R123L-R127Q-R179Q/0.5 ppm	6.1±1.2	6.8±2.1	0.4±4.1	1.0±1.4

In another group experiment, tested the soil release ability of multiple multiple replacement ASP variant.Use test condition mentioned above, wherein carry out following modification.In these tests, by with 37.5gm

2005 SNOW are dissolved in 25 liters of 105ppm water with the preparation of 105ppm water 2005SNOW detergent solution (1.5gm/L).Then add 30g Hepes (=5mM; C ₈H ₁₈N ₂O ₄S), stirred solution, and with ± 19ml 4N NaOH with pH regulator to 7.8.Notice that this solution can at room temperature preserve 36 hours.In these tests, the sample that only uses sample that EMPA 116 BMI of 10 * 7.5cm make dirty and 10 * 7.5cm Equest grass medium to make dirty.A kind of concentration determination variant with 0.55ppm.In each beaker, add 6 samples (6

EMPA

116 or 6 samples that Equest grass medium is made dirty), add 1 at every turn.In addition, add 2 EMPA 221 samples.Use do not add any enzyme sample contain the HEPES buffer reagent 2005 SNOW detergent solution (Procter ﹠amp; Gamble) in contrast.Each scourability test is all carried out in quadruplicate.Result's (average delta %SR, standard deviation (STD) and repeat number (n)) is shown in Fig. 7.

Embodiment 11

The soil release performance of the multiple replacement mutant of under low pH, testing

The experiment of the soil release performance that is used to measure multiple multiple replacement ASP variant has been described in the present embodiment.In these experiments, use embodiment 1 described scheme as mentioned.The variant (comprising the sudden change in every kind of variant) that following table provides activity to be higher than wild-type ASP+1 times standard deviation (1.1 performance index).

In other experiments of using liquid washing agent, obtained following result.The variant that marks in the following table shows the activity that is higher than wild-type+1 times standard deviation (＞1.1 performance index).

Embodiment 12

The clean-up performance of ASP variant in automatic bowl

Carried out experiment in the present embodiment to measure the ASP variant at automatic bowl (AutomaticDish Washing, ADW) TABLET ACTION

Compare the clean-up performance of described proteolytic enzyme susceptibility spot in the washing composition with benchmark serine protease (" proteolytic enzyme B ").As shown in the table, the soil release of ASP variant is more much better than proteolytic enzyme B.

Carried out the micro-washing test that carries out with 24 orifice plates.Get the stainless steel plate with hammer and staking punch.Plate is cleaned and weighed, to obtain starting weight.(" the egg prepared product of stirring " and " yolk prepared product ") as mentioned below preparation egg spot.Then the egg that 50 μ L are prepared is distributed on each plate.Made prepared product at room temperature dry 30 minutes, and then in baking oven, toasted 2 hours with 80 ℃.Plate is cooled to room temperature.After making dirty, plate is weighed to obtain the spot weight on the plate.The plate that to make dirty inserts

In the 24 hole polystyrene flat boards.By thorough mixing prepares the automatic bowl product solution in 1L 11gpg water under 40 ℃ the ADW of appropriate amount not being had enzyme product (for example powder 4500ppm).By with 188.57g CaCl ₂2H ₂O and 86.92g MgCl ₂6H ₂O is mixed in the 1L DI water and prepares hardness solution.In these tests, at the ADW solution that in each hole, adds the 2mL preheating under 55 ℃.Then add an amount of enzyme to each hole.In some embodiments, test uses 6 intersection (across), 4 to repeat to carry out, and in other embodiments, tests with 4 intersections and 6 and repeats to carry out.Then use the film phonograph seal flat board.Flat board is placed the incubator/shaking table of preheating and uses the box clamping fixed.Flat board (being 55 ℃ for European wash conditions for example) under suitable temperature was washed 30 minutes with 180RPM.After the washing, flat board is immersed in the warm water bath 3 times carefully carry out rinsing.Plate is taken out from flat board, and in baking oven with 80 ℃ of dryings 1 hour.In case after the drying, plate is weighed with the plate weight after the acquisition washing.Following calculated weight assessment draws proximate removing per-cent:

% removes=(the back weight-weight after washing of making dirty)/(the back weight-cleaning weight of making dirty) * 100

Remove the removing of index=% removing/% benchmark proteolytic enzyme (proteolytic enzyme B)

The egg prepared product that stirs

Be prepared as follows the egg that is used for the test proteins enzyme and/or comprises the detergent composition of tested enzyme.At first, the 100ml10% whole-milk is mixed with 3 complete eggs.Boil this mixture and constantly stirring, a little easily flow until this mixture.Then add 40ml milk again, and by hand mixer or agitator with the mixture mixing, until smooth and do not have an agglomerate sign.Before making dirty egg mixture is cooled to room temperature.

The yolk prepared product

By being arranged to 60 ℃, the hygrosensor on the backing comes heated water bath.On beaker, place the slurry strainer.In order to prepare yolk, 6 to 9 eggs are smashed, yolk and albumen sepn.Eggshell is not broken, but removes any residue that exists in the yolk.With yolk soft rinsing in cold water.Yolk is smashed on strainer and beaker lightly.In case all yolk all filter, the beaker that just will contain filterable yolk places water-bath.Yolk was stirred 3 minutes down at 60 ℃.From water-bath, take out yolk, and placed cooling bath 30 minutes.Remove the surface lightly and go up any egg skin that forms.

Use stainless steel substrates (1 * 3 inch) preparation egg spot

Immerse each tinsel in the egg that stirs or the yolk prepared product (as above-mentioned preparation) and pat once, guarantee to have on each sheet the egg of roughly the same amount.With the back side of sheet on paper handkerchief wiping so that its cleaning.Then sheet is placed on two oven frames by numerical order.Made sheet at room temperature dry 30 minutes.Then sheet was boiled 2 hours exchange upper strata frame and lower floor's frame after 1 hour, and Rotate 180 ° (+/-1 °) under 80 ℃ (+/-1 ℃).The dirty sheet of refrigerative is weighed with the order of 1-96.

For washing, 8 spot sheets are placed on the upper strata frame and lower floor's frame of washing machine.Pre-wash and main washing are based on the capacity of 840 glasss of Whirlpool, and pre-wash is 40ml, and main washing is 60ml.Generally in a test, with 4 products of four GE 500 machines operations.With regard to test loop, with pack into machine and test is placed the product of pre-wash and main washing cup of dirty sheet.Water temperature is set to 120 °F, and machine moves with normal cycles of washing.Taking out sheet when final rinse cycle finishes from machine is used to weigh.These sheets do not experience dry recycle.When repeating to finish for the 4th time, each product moves once in each machine.Repeat all in one day, to carry out for all four times.Second day, use new dirty matrix to repeat identical step, so that being provided, each product amounts to 8 repetitions, the then average net result that obtains.Following calculated weight analysis and evaluation draws the approximate per-cent of removing:

% removes=(weight after making dirty-weight after washing)/(weight after making dirty-cleaning weight) * 100

Hereinafter show to provide among the 12-1 result of these performance tests:

Embodiment 13

The clean-up performance of ASP variant in the powder laundry detergent

In the present embodiment, described and be used to measure the ASP variant at the laundry powder washing composition

In the experiment of clean-up performance.In these experiments, the performance of variant and benchmark serine protease (for example " proteolytic enzyme C ") are cleaned at material object (Real Item Cleaning compares in RIC).

Use the Meile washing machine to test: 40 ℃ of temperature, hardness 21gpg, washing composition 7300ppm, proteinase-10 .88ppm, RI 0.75kg, total ballast weight and RI 2.5kg, water 13L, washing time 20 minutes, 3 * 5 minutes rinsing time with following condition.

In these experiments, with the sheet of king size and queen size and clean socks (J﹠amp; RCoordinating Service) quartern is as clean ballast weight.In addition, to human consumer's material object (Consumer Real Items RI) tests, and comprises socks, T-shirt, pillowcase, towel and tea cloth).Use in these experiments and comprise yeast and AS1 (artificial spot 1; Equest) spot.The composition of AS1 is shown to provide among the 13-1 hereinafter.

Clothing is cut off to guarantee that the clothes size that will equate is used for test, is chosen to uniform spot level with clothing.With the pairing in form order to two halves encode (for example left/right of clothing).

Clothing 1 clothing 2 clothings 3 clothings 4 clothings 5 clothings 6 clothings 7 clothings 8

AB BA BA AB BA AB AB BA

The clothing that will be labeled as " A " washs with the ASP variant, and is labeled as the clothing proteolytic enzyme C washing of " B ".Test is carried out (each is handled in four machines and repeats four times, and the inner repetition of two RI is wherein arranged) with 4 * 4 * 2.

Testing method

With clean quartern sheet ballast weight and RI combination,, and in the manual Meile washing machine of packing into, open " 21gpg " water-in on the front panel with generation gross weight 2.5kg.Then open washing machine.Temperature is arranged to 40 ℃ and short cycles of washing.Then, ballast weight and RI are added in the washing machine.In addition, the clean socks that in each repeats, add 20g AS1 and make dirty with the 17g bread yeast.Then in the washing composition pallet of washing machine, add washing composition (95g) and begin circulation.In case after washing composition is flushed away, just add any liquid to be added from pallet.Then machine moves its circulation.When cycles of washing is finished, inclusion was transferred in the drying machine dry 30 minutes.Then article are graded, so that the preference number in PSU result and the LSG program to be provided by range estimation.

The test grading

During single in washing and dry test article 72 hours whole test is graded.The article clothing that separates is layered in the Meeting Room, and grades according to Scheff é yardstick by at least 3 judges.In addition, spot A and B are at least by 2 judge's gradings.

Result treatment

The grading input is installed the portable Psion of washing test software.When finishing the grading of all separate items, data are transferred in the Personal Computer, wherein calculate the mean P SU classification, A of every clothing and B preference % and the LSD between handling.With the significant difference between 90% the fiducial limit counting yield.

Use the PSU rating system to come two kinds of products of comparison (prescription).Two kinds of prescriptions are tested (for example the spot after the washing is residual) to performance.In these experiments, some fabrics that will wash with two kinds of products (for example product A and product B) simultaneously compare.Grade by two or more several judge, wherein use following Schelle yardstick:

0: no preference

1: I feel this product good slightly (uncertain)

2: I know that this product is good slightly

3: this product is better

4: this product is much better.

Embodiment 14

The storage stability of ASP variant

In the present embodiment, described and be used to measure liquid The experiment of the storage stability that the multiple ASP variant in the washing composition is compared with WT ASP.Use the protease activity of Hitachi 911 automatic analysers test variant and WT ASP.In these tests, use succinyl--L-Ala-L-Pro-L-Phe-that p-nitroanilide (pNA) is come the evaluating protein enzymic activity as substrate.In this test, the Carboxylamide key by the active protease incision tip obtains p-Nitroaniline.Protease activity under the 415nm/450nm in gained xanchromatic intensity and the sample is proportional, with

The calibration of proteolytic enzyme standard substance.

As shown in following table, compare the remarkable storage stability that improved with WT ASP through the variant of transforming.In this table, use following formula to measure " retentive activity % ": retentive activity %=activity/original activity * 100%.

Embodiment 15

Measure the cleaning action of ASP

In the present embodiment, the experiment that is used to measure the cleaning action of ASP under multiple condition has been described, and the characteristic of described multiple wash conditions.

There is multiple wash conditions, comprises multiple detergent formulation, washing water volume, temperature of washing water and washing duration.Therefore, detergent component (as proteolytic enzyme) must be able to tolerate these unsuitable environmental conditions and bring into play function.For example, be used for the concentration difference of the detergent formulation of different areas at its relevant composition of washing water.For example European washing composition generally contains the detergent ingredients of the 3000-8000ppm that has an appointment in washing water, and Japanese washing composition generally contains the detergent ingredients that is lower than 800 (for example 667ppm) in washing water.Particularly in the U.S., washing composition generally contains the detergent ingredients of have an appointment 800 to 2000 (for example 975ppm) in washing water in the North America.

The Latin America washing composition generally contains high bubble phosphoric acid buider washing composition, and the detergent range that use in Latin America can fall into the high detergent concentration scope of neutralization, because they contain the detergent ingredients of 1500ppm to 6000ppm in washing water.Brazil's washing composition generally contains the detergent ingredients of the 1500ppm that has an appointment in washing water.Yet other high bubble phosphoric acid buider washing composition areas (being not limited only to other Latin American countries) can have the high detergent concentration system that contains up to about 6000ppm detergent ingredients in washing water.

In view of above content, obviously, detergent composition concentration in typical case's washing soln is worldwide from being lower than about 800ppm detergent composition (" low detergent concentration area ", Ri Ben 667ppm for example) to about 800ppm about 2000ppm (" middle detergent concentration area " extremely, the 1500ppm of about 975ppm of the U.S. and Brazil for example) do not wait to being higher than about 2000ppm (" high detergent concentration area ", for example Ou Zhou about 3000ppm extremely about 8000ppm and height steep the geographic about 6000ppm of phosphoric acid buider).

The concentration of typical case's washing soln is rule of thumb determined.For example, in the U.S., typical washing machine holds the washing soln of about 64.4L volume.Therefore, in order to obtain the detergent concentration of about 975ppm in the washing soln, must in about 64.4L washing soln, add about 62.79g detergent composition.This amount is that the human consumer is provided by the typical amount in the adding washing water that the measuring cup that provides with washing composition weighs.

Again for example, different wash temperatures is used in different areas.The temperature of washing water of Japan generally is lower than Europe.For example, the temperature of washing water of North America and Japan can be 10 to 30 ℃ (for example about 20 ℃), and the temperature of European washing water is generally 30 to 50 ℃ (for example about 40 ℃).

Again for example, different areas generally have the different water hardness.The water hardness is generally by per gallon mixed C a ²⁺/ Mg ²⁺In grain (grain) number describe.Hardness is calcium (Ca in the water ²⁺) and magnesium (Mg ²⁺) amount tolerance.Most water of the U.S. are hard, but hardness is different between different areas.Medium hardness water (60-120ppm) to hard water (121-181ppm) has the hardness material of 60 to 181ppm (ppm is converted to grain/US gallon: the ppm number is grain/gallon divided by 17.1).Table 15-1 provides the scope of the water hardness.

The water hardness in Europe generally is higher than 10.5 (for example 10.5-20.0) grain/gallon mixed C a ²⁺/ Mg ²⁺(for example about 15 grains/gallon mixed C a ²⁺/ Mg ²⁺).The water hardness of North America generally is higher than the water hardness of Japan, but is lower than the water hardness in Europe.For example, the North America water hardness can be 3 to 10 grains, 3 to 8 grains or about 6 grains.The water hardness of Japan generally is lower than the water hardness of North America, is usually less than 4, for example 3 grains/gallon mixed C a ²⁺/ Mg ²⁺

The invention provides the ease variants that has the scourability of raising in (usually under multiple wash conditions) under at least one group of wash conditions.

As described herein, use little sample determination method test proteins enzyme variants in dissimilar washing composition and the performance in the wash conditions (seeing above and U.S. Patent Application Serial Number 09/554,992 and WO99/34011, all incorporated herein by reference).Also in a similar fashion to other spot substrate test proteins enzyme variants.

Embodiment 16

The liquid fabric cleaning compositions

Present embodiment provides and can be used for clean fabric composition of the present invention.Think that these compositions are under Japan's machine washing condition and comprise in the application of the meticulous and/or fragile fabric of cleaning particularly useful.Table 13-1 provide suitable composition.Yet the present invention also is not intended to and only limits to this special formulation, and many other prescriptions all can be used for the present invention.

The pH of embodiment 16-4, composition (I)-(II) be about 5 to about 7,1 (III)-(V) be about 7.5 to about 8.5.#1: add the 1N HCl aqueous solution with the clean pH regulator of preparation to about 3 to about 5.

Embodiment 17

The liquid composition that washes the dishes

Present embodiment provides and can be used for the liquid of the present invention cleaning compositions that washes the dishes.Think that these compositions are particularly useful under Japan washes the dishes condition.Yet the present invention also is not intended to and only limits to this special formulation, and many other prescriptions all can be used for the present invention.

The invention provides the high-density dishwashing detergent composition of following densification.

^*Whitening agent/dyestuff/SRP1/ Xylo-Mucine/optical white/MgSO ₄/ PVPVI/ defoamer/high molecular weight PEGs/clay.

The pH of composition among the embodiment 17-1 (I) to (VI) is about 9.6 to 11.3.

The invention provides the following hand washing liquid detergent composition that washes the dishes.

The invention provides following liquid automatic dishwashing detergents composition.

The invention provides following tablet detergent composition.These compositions change squeezing machine with 13KN/cm by use standard 12 great wheels ²Pressure compressed granulate shape dishwashing detergent composition prepare.

The pH of composition among the embodiment 17-4 (I) to (VIII) is about 10 to about 11.5.

The tablet of (I) to (VIII) heavily is that about 20 grams are to about 30 grams among the embodiment 17-4.

The composition that below washes the dishes is particularly useful under Japanese wash conditions.

Embodiment 18

The liquid fabric cleaning compositions

Proteolytic enzyme of the present invention is particularly useful in cleaning compositions.For example, consider produced according to the present invention under Japan's machine washing condition useful especially liquid fabric cleaning compositions.In some preferred embodiments, these compositions contain the following composition shown in the table 18-1.

Embodiment 19

Particulate state clean fabric composition

In the present embodiment, provide the multiple particulate state clean fabric composition of the present invention that can be used for.Following table provides suitable composition.Yet the present invention also is not intended to and only limits to these concrete prescriptions, and many other prescriptions all can be used for the present invention.

The invention provides following laundry composition.These compositions are suitable for using as particle or tablet.

^*Spices/dyestuff, whitening agent/SRP1/ Xylo-Mucine/optical white/MgSO ₄/ PVPVI/ defoamer/high molecular weight PEGs/clay.

Following laundry detergent composition is considered particularly useful under the machine washing condition of Europe.

Embodiment 20

The hard-surface cleaning detergent composition

The present invention also provides the composition that is applicable to cleaning of hard surfaces.

The pH of these compositions is about 7.4 to about 9.5.

Embodiment 21

The animal-feed that comprises ASP

The present invention also provides the animal feedstuff compositions that comprises the ASP variant.In the present embodiment, provide so a kind of feed that is applicable to poultry.Yet the present invention also is not intended to and only limits to this concrete prescription, and proteolytic enzyme of the present invention can be used in multiple other feed formulations.Feed of the present invention also is intended to be suitable for any animal is used, and includes but not limited to domestic animal (for example ox, pig, sheep etc.) and companion animals (for example dog, cat, horse, rodent etc.).Following table provides and has been suitable for mashed prod that the turkey poult in age in height to 3 week is used, promptly based on the prescription of the initial feed of corn.

In some embodiments, in this feed formulation, replenish proteolytic enzyme of the present invention (for example 2000 units/kg, 4000 units/kg and 6000 units/kg) of multiple concentration.

All patents mentioned in this specification sheets and publication are all represented one of ordinary skill in the art's of the present invention level.All patents and publication are all incorporated herein by reference, and its degree all indicates incorporated herein by reference separately as every piece of publication particularly.

Described after the preferred embodiments of the invention, it will be apparent to those skilled in the art that and can carry out multiple modification to disclosed embodiment, these modifications all are intended to be included among the scope of the present invention.

Those skilled in the art are easy to recognize that the present invention is suitable for implementing purpose of the present invention and obtains described result and advantage and intrinsic result and advantage.Composition described herein and method are represented preferred embodiment, for illustrative, are not intended to limit the scope of the invention.It is apparent that for a person skilled in the art, can carry out a variety of substitutions and modifications to invention disclosed herein, and not depart from scope and spirit of the present invention.

The invention that this paper illustrative is described can suitably be implemented under the situation that lacks not concrete disclosed any key element of this paper or restriction.Employed term and statement are used for describing and unrestricted, and the use of these terms and statement is not intended to get rid of any equivalent or its part of described feature, but should be realized that, in the claimed scope of the invention multiple modification can be arranged.Therefore, should be appreciated that, although specifically disclose the present invention by preferred embodiment and optional feature, those skilled in the art can use the modification and the change of notion disclosed herein, and these modifications and change are all thought within the scope of the invention that limits at appended claims.

This paper is wide in range and described the present invention prevailingly.Each the narrower scope and the refinement grouping that fall within general the disclosing also constitute a part of the present invention.This comprises that to general description the of the present invention, its prerequisite or reverse side are restricted to and remove any theme from such, and whether the content of no matter being removed specifically describes in this article.

Claims

1. isolating serine protease variants, it has and comprises the aminoacid sequence that at least two amino acid are replaced, and carries out on the position of wherein said replacement position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

2. the composition that comprises the isolating serine protease variants of claim 1.

3. the composition that comprises the isolating serine protease variants of claim 1, the Serine Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence has the immunology cross reactivity shown in wherein said isolating serine protease variants and the described SEQ of the comprising ID NO:8.

4. the isolating serine protease of claim 1, wherein said sequence comprise at least two amino acid to be replaced, and wherein said at least two amino acid positions are selected from 1,2, and 3,4,7,8,9,10,11,12,13,14,15,16,18,19,22,24,25,26,27,28,29,30,31,32,33,34,35,36,38,39,40,41,42,43,44,45,46,47,48,49,51,52,54,55,56,57,59,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,96,99,100,101,103,104,105,107,109,110,111,112,113,114,115,116,117,118,119,121,123,124,125,126,127,128,129,130,132,133,134,135,136,137,140,141,142,143,144,145,146,147,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,172,175,176,177,179,180,181,182,183,184,185,186,187,188 and 189, carry out on the position of wherein said at least two replacements position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ IDNO:8.

5. the isolating serine protease of claim 4, wherein said proteolytic enzyme comprise and are selected from least two following replacements: G12D, R14I, R14L, R14M, R14S, R16I, R16L, R16Q, N24E, N24H, N24M, N24T, N24W, R35E, R35F, R35H, T36S, G49A, G54D, G54L, R61V, A64K, G65Q, Q71F, Y75G, S76A, S76L, S76N, S76T, S76V, R79K, R79T, Q81K, Q81P, T86K, A93G, A93H, A93S, S99A, T109M, N112E, T116E, R123F, R123L, R123Q, R123S, R127A, R127K, R127Q, R159E, R159F, R159G, R159K, R159L, R159Q, R179N, R179Q, I181K, I181Q, I181T, D184N, D184T and S187Q carry out on the position of wherein said two replacements position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8 at least.

6. the proteolytic enzyme of claim 5, wherein said proteolytic enzyme comprises and is selected from following multiple replacement: R16Q/R35F/R159Q, R16Q/R123L, R14L/R127Q/R159Q, R14L/R179Q, R123L/R127Q/R179Q, R16Q/R79T/R127Q, R16Q/R79T, R35E/R123L/R127Q/R179Q and G12D/R35E/G63R/R79K/T109M.

7. the proteolytic enzyme of claim 5, wherein said proteolytic enzyme comprises following replacement: R123L, R127Q and R179Q.

8. the proteolytic enzyme of claim 4 comprises in the aminoacid sequence of wherein said proteolytic enzyme and is selected from least two following replacements: D2G, D2Q, V3I, V3L, N7A, N7L, N7S, I11A, I11Q, R14E, N24A, N24E, N24H, N24L, N24M, N24Q, N24T, N24V, T36D, T36F, T36G, T36H, T36I, T36L, T36N, T36P, T36R, T36S, T36V, T36W, T36Y, A38D, A38F, A38H, A38L, A38N, A38R, A38S, G49F, S51A, G54A, G54D, G54H, G54K, G54L, G54M, G54R, N55F, A64H, A64N, A64R, A64W, A64Y, G65L, G65P, G65Q, G65R, G65S, G65T, G65Y, G65V, V66A, V66D, V66E, V66H, V66I, V66L, N67A, N67G, N67L, N67K, L69H, L69S, L69V, A70D, A70H, A70S, Q71A, Q71G, Q71H, Q71I, Q71K, Q71M, Q71N, N73S, N73T, N74G, Y75F, Y75G, Y75I, S76L, S76Y, S76V, S76W, G77S, G77T, G78A, G78D, G78H, G78N, G78S, G78T, R79P, V80H, V80L, Q81H, Q81K, Q81V, H85Q, H85T, V90I, V90P, V90S, S92G, W103I, W103M, H104K, T109A, T109H, T109I, S114G, T116F, P118A, P118F, P118H, P118R, E119K, E119R, N145E, N145I, N145Q, V150L, R159F, N170Y, G177M, R179A, R179D, R179E, R179I, R179K, R179L, R179M, R179N, R179T, R179Y, R179V, I181H, T183I, G186E, G186I, G186V, S187P, S187T and S188M.

9. the proteolytic enzyme of claim 4 comprises in the aminoacid sequence of wherein said proteolytic enzyme and is selected from least two following replacements: F1A, F1T, D2A, D2H, D2N, V3T, N7H, N7I, A8G, A8K, T10G, T10K, I11S, I11T, G12W, G13M, S15F, N24F, N24S, A30S, R35F, T36C, A38G, A38I, A38K, A38V, A38Y, T40S, T40V, A41N, N42H, F47I, F47M, G49A, G49K, G49L, S51F, S51Q, G54I, G54Q, N55K, N55Q, R61M, T62I, G63Q, G63V, G63W, A64F, A64I, A64K, A64L, A64M, A64Q, A64S, A64T, A64V, G65A, G65H, V66M, V66N, N67D, N67F, N67H, N67Q, N67R, N67S, N67T, N67V, N67Y, L68W, L69W, A70G, Q71D, Q71F, Q71L, Q71R, V72I, N73H, S76E, S76I, S76K, S76A, S76N, S76Q, S76R, S76T, G77N, G77Y, G78I, S78R, G78V, R79G, V80F, Q81D, Q81I, A83N, H85R, H85K, H85L, T86A, P89N, V90A, V90L, V90T, T107H, T107M, T107S, T107V, T109G, T109L, T109P, T109R, A110S, A110T, N112I, P118E, P118I, P118K, P118Q, R123E, R123I, I126L, R127F, I28L, T129S, E133Q, L142V, A143N, A143S, N145G, N145L, N145T, V150M, T151L, R159E, T163L, Q167N, N170A, N170D, N170L, 171S, G177S, I181G, I181N, T182V, T183K, T183M, D184F, D184H, D184Q, D184R, S185I, S185V, S187E and S187L.

10. the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme comprise and are selected from least two following replacements: D2P, A8G, T10C, T10L, I11E, I11Q, I11T, I11W, G12D, G12I, G12N, G12Q, G12S, G12V, R14A, R14C, R14E, R14D, R14G, R14I, R14N, R14Q, R14S, R14T, S15C, S15E, S15H, S15R, S15Y, R16C, R16D, R16E, R16T, R16V, A22C, A22S, N24E, R35A, R35C, R35D, R35E, R35H, R35M, R35N, R35P, R35Q, R35S, R35T, R35V, T36C, A38C, A38D, A41C, A41D, T44E, T46C, T46E, T46F, T46V, T46Y, F47R, A48E, G49A, G49C, G49E, G49H, G49L, G49N, G49Q, G49V, G54C, N55G, D56L, Y57G, F59W, R61E, R61M, R61T, R61V, G91Q, S99A, T100A, T100R, T107R, T109E, N112P, S113C, S114C, P118K, P118R, E119G, E119R, E119T, E119V, E119Y, T121E, T121F, T121L, R123C, R123D, R123E, R123F, R123H, R123N, R123Q, R123S, R123T, R123V, R123W, R123Y, G124D, L125Q, R127D, R127E, R127K, R127Q, R127S, P134R, T151C, T151L, S155C, S155I, S155W, S155Y, R159D, R159E, R159Q, R159S, R159T, R159V, T163D, F165E, F165W, Q167E, N170C, N170D, G177D, R179D, R179E and M180L.

11. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: F1N, F1P, D2I, D2M, D2T, D2V, A8R, A8T, T10D, T10E, T10F, T10M, T10Q, T10Y, G12H, G12P, G12Y, G13D, G13E, R14H, R14L, R14M, S15F, S15G, S15N, R16I, R16Q, N24G, N24T, I28V, R35K, T36V, A41S, A41T, N42D, T44C, F47V, G49F, G49K, G49S, S51A, S51C, S51L, S15M, G54E, N55A, D56F, R61K, R61Q, A64C, G65D, V66N, A70G, A70M, A70P, R79T, R79V, Q81A, Q81G, Q81P, A83E, A83D, A83H, T86E, A87C, A87E, A88F, S92T, S99G, S99H, S99K, S99Q, T100K, T100Q, W103L, T109K, N112D, N112E, S113A, S113D, S114E, T115C, T116G, T116N, P118A, P118C, P118G, P118W, E119A, E119L, E119N, E119Q, E119S, T121A, T121D, R123A, R123G, R123I, R123K, R123M, A132S, L125M, R127A, R127C, T128A, S140P, L141M, T151V, S155E, S155F, S155T, S155V, N157D, R159A, R159C, R159K, R159M, R159N, T160D, T163C, F165H, N170L, I172A, Q174C, Q174S, Q174T, A175T, G177E, R179C, R179F, R179I, R179L, R179M, R179N, R179S, R179T, R179V, R179W, R179Y, S187E and S188E.

12. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: V3R, I4D, I4G, I4P, Y9E, Y9P, T10F, T10W, T10Y, G12D, S18E, A22C, A22S, A22T, N24T, G26E, G26I, G26K, G26Q, G26V, G26W, F27V, F27W, I28P, I28T, T29E, A30M, A30N, A30P, A30Y, G31H, G31M, G31N, G31V, G31Y, C33E, C33L, C33M, C33N, A38D, A38G, T39R, T40D, T40H, T40N, T40P, T40Q, R43D, P43G, P43H, P43K, P43L, P43N, G45A, G45V, T46V, T46Y, T46W, A48P, Y57M, Y57N, F59K, T62G, T62R, A70G, A70P, N73P, R79T, Q81A, Q81D, Q81F, Q81G, Q81H, Q81P, Q81S, A83H, G84C, G84P, P89W, G91L, A93S, R96C, R96E, R96F, S99A, T100A, C105E, C105G, C105K, C105M, C105N, C105P, C105S, C105W, T121E, R123F, R123N, R123W, R123Y, L125A, T128A, T128C, T128G, T128S, T128V, I28W, T129W, S137R, S140P, Q146P, A147E, S155F, S155K, S155P, S155R, S155W, S155Y, G156I, G156L, G156P, C158G, C158H, C158M, R159K, T160I, G161I, G161L, G161V, T164G, T164L, F166S, Q167L, P168Y, Y176P, G186S and S188A.

13. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: A8G, T10C, T10L, G12A, G12H, S15C, S15N, S15Q, S15R, S15T, N24E, N24S, G25S, F27I, G31A, H32A, C33D, T36V, T39V, A41S, T46F, G49A, S51V, F59W, Q71A, Q71Y, N74F, R79V, Q81C, Q81E, A83E, A83F, A83M, A83R, G84M, G84V, T86I, T86M, T86S, A87E, A87S, P89A, V90A, V90M, S92T, A93D, S99G, T100Q, T101S, W103N, C105A, C105L, C105T, C105Y, T107A, T107F, T107L, T107Q, T107S, T110D, A110G, L111K, V115I, V115L, T116Q, Y117K, Y117Q, Y117R, Y117V, P118T, E119L, T121A, T121D, R123I, R123K, R123L, R123Q, R123T, L125M, R127F, R127K, R127Q, T129Y, V130T, A132C, P134W, L141C, A143H, G144A, V150N, T151C, G153K, G153V, G154L, G154R, S155T, R159Q, R159T, R159V, T160E, T160Q, G161K, G162P, T163I, F166A, F166C, P168I, N170D, N170E, G177N, R179K, M180L, T182L, T183A, T183I, T183P, S185R, G186P, S188C, S188E, S188G, S188M, S188T, S188V and P189S.

14. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: F1T, T10N, R14D, R14G, R14I, R14L, R14N, R14Q, R14T, N24A, N24E, N24H, N24L, N24Q, N24T, N24V, R35A, R35E, R35F, R35L, R35Q, R35T, T36G, T36I, T36N, T36S, A38D, A38F, A38H, A38N, A38R, G49A, G49S, S51D, G54D, G54E, N55E, N55F, A64I, G65D, G65P, G65Q, G65S, G65T, G65V, N67D, L69S, N73T, N74G, Y75F, Y75G, S76D, S76E, S76I, S76L, S76N, S76T, S76V, S76Y, G77T, G78A, G78D, R79A, R79D, R79E, R79G, R79L, R79M, R79P, R79S, R79T, R79V, Q81E, A83E, H85Q, H85T, T86D, T86E, V90I, V90P, V90S, V90T, S99N, S99V, T107E, T107H, T107S, T107V, T109E, N112D, N112E, N112L, N112Q, N112V, T116E, T116Q, T121E, R123A, R123D, R123E, R123F, R123H, R123I, R123L, R123N, R123Q, R127A, R127Q, T129S, L142V, N145E, R159D, R159E, R159F, R159N, R159Q, N170D, N170Y, I172T, R179A, R179D, R179E, R179I, R179K, R179M, R179N, R179T, R179V, R179Y, I181L and G186N.

15. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: F1D, V3L, N7L, A8E, A8G, T10D, T10E, G12D, G13S, R14A, R14K, R14S, R14M, S15W, I19V, N24M, R35H, R35M, R35S, R35W, R35Y, T36D, T36H, A38L, A38S, A38T, A38Y, T40V, A41D, A41N, A48E, G49F, G49H, S51H, S51Q, S51T, S51V, R61E, R61H, R61M, R61S, R61T, G63D, A64F, A64H, A64L, A64M, A64N, A64P, A64Q, A64S, A64T, A64V, A64W, A64Y, G65L, G65Y, N67E, N67G, N67H, N67S, N67T, A70D, A70G, A70H, Q71D, Q71G, Q71H, Q71S, V72I, S76Q, S76W, G77S, Q81D, Q81H, Q81V, H85L, H85M, V90N, S92A, S92G, A93D, A93E, A93S, S99D, S99T, T101S, W103M, T107A, T107I, T107M, T107N, T109A, T109G, T109I, A110S, N112Y, S113T, S114A, V115A, T116F, T121D, N121I, R123G, R123S, R123T, R123V, R123Y, R127H, R127K, R127E, R127S, R127Y, N145D, N145T, R159A, R159C, R159K, R159L, R159S, R159Y, T160E, T163D, N170L, R179L, T182V, T183E, T183I and S185N.

16. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: D2Q, V3L, N7L, I11A, I11Q, R14I, R14M, R16L, R16Q, N24A, N24E, N24H, N24M, N24Q, N24T, N24V, R35F, R35L, T36D, T36G, T36H, T36I, T36L, T36N, T36P, T36S, T36W, T36Y, A38L, A38R, A38S, A48Q, G49A, G54D, G54I, G54Q, G54N, R61V, A64F, A64H, A64Y, G65L, G65P, G65Q, G65S, G65T, G65Y, V66H, N67A, N67G, N67L, N67S, N67V, N67Y, L69H, L69S, Q71I, N73T, N74G, Y75F, Y75G, Y75I, S76A, S76D, S76E, S76I, S76L, S76N, S76T, S76V, S76W, S76Y, G77T, G78D, R79G, R79P, Q81P, H85F, H85K, H85L, H85Q, H85R, P89D, S92A, A93T, A93S, S99A, S99D, S99N, S99T, S99W, T109E, N112E, S113A, S114G, T116F, T121D, R123F, R123I, R123L, R127A, R127F, R127G, R127H, R127K, R127L, R127Q, R127S, R127T, R127Y, A132V, P134E, A143N, N157D, R159D, R159E, R159F, R159H, R159K, R159N, R159Y, G161K, N170Y, R179V, I181Q, D184F, D184H, G186E, G186I, G186V and S187P.

17. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: F1T, A8D, A8G, T10E, T10L, T10Q, I11L, I11S, I11T, G12D, G12Y, R14E, R14L, R14N, R14P, S15E, R16A, R16G, R16I, R16N, N24L, N24S, V31F, R35A, A38D, A38F, A38N, A38V, A38Y, T40V, A41N, N42H, G49F, G49H, G49S, S51Q, S51T, G54A, G54L, G54M, N55F, R61H, R61K, R61M, R61S, R61T, A64N, A64S, A64T, A64V, A64W, G65R, G65V, V66D, N67F, N67K, N67M, N67Q, N67T, L69W, A70G, A70P, Q71D, Q71F, Q71H, Q71L, Q71T, G77N, G77S, G78A, G78N, R79D, V80H, V80L, H85T, H85Y, T86N, A88F, P89N, P89V, V90I, V90P, V90T S92G, A93D, A93E, S99G, L111D, L111E, N112D, N112G, N112L, N112Q, S113G, T121E, R123E, R123K, R123Q, L125V, P134G, S140A, L142V, A143S, N145D, V150L, R159A, R159C, R159L, R159V, T160E, G161E, T163D, T163I, N170D, N170L, R179D, R179E, R179K, R179N, R179T, I181H, T183I, D184R, D184L, D184Q, D184T, S185W, S185I, G186L, S187E, S187Q and S188Q.

18. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: A8R, A8S, A8T, A8V, R14E, R14L, R14M, R16Q, N24A, N24E, N24Q, N24T, R35F, R35L, T36D, T36G, T36I, T36N, T36P, T36S, A38D, A38F, A38L, A38R, A38S, S51A, S51D, G54D, G54I, N55E, N55F, N55S, R61M, R61T, G63V, A64H, A64N, A64S, G65Q, G65P, G65R, G65S, G65T, G65Y, V66D, N67D, S76E, N67F, N67G, N67L, N67M, N67S, N67T, N67V, N67Y, L69H, L69S, L69V, L69W, N73T, N74G, Y75F, Y75G, S76C, S76D, S76I, S76L, S76N, S76W, S76Y, S76V, G77T, G78D, R79C, R79D, R79E, R79G, R79P, Q81V, A83N, T85A, H85Q, T86F, T86I, T86L, V90I, V90N, V90P, V90S, V90T, A93D, A93E, T107M, T107N, T107S, T109A, T109E, T109I, N112E, T121D, T121E, R123D, R123E, R123F, R123I, I126L, R127A, R127H, R127K, R127L, R127Q, R127S, R127Y, P134A, P134E, L142V, A143N, N145E, N145S, R150Y, R159C, R159D, R159E, R159F, R159K, R159Q, G161E, T163D, N170Y, I172V, G177M, R179A, R179D, R179E, R179I, R179K, R179L, R179M, R179N, R179T, R179V, R179Y, M180D, T182V, T183I, G186E, G186V and S187P.

19. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: V3L, I4M, A8E, A8H, A8L, A8N, A8P, I11T, R14I, R14Q, R16L, N24H, N24L, N24M, N24V, R35A, R35E, T36V, T36Y, A38H, A38I, A38N, T40V, A41N, G49A, G49L, S51F, S51Q, G54A, G54E, G54M, G54Q, N55Q, N55V, R61V, T62I, G63D, G63L, G63P, G63Q, A64F, A64I, A64L, A64M, A64Q, A64R, A64T, A64V, A64W, G65A, G65D, V66E, N67A, N67C, N67Q, N67R, L69Q, A70G, A70P, A70S, Q71D, Q71M, S76A, S76Q, S76T, G77N, G77Q, R79L, Q81E, Q81H, Q81I, A83D, A83I, H85L, H85R, T86E, T86M, A88F, V90L, S92C, S92G, A93Q, R96K, T101S, W103M, W103Y, T107A, T107E, T107H, T107Q, T107V, T109G, T109H, T109L, T109N, A110S, A110T, N112D, S114G, T116F, T121L, R123A, R123H, R123K, R123L, R123P, R123Q, L125V, R127F, R127T, T129G, T129S, A132V, P134D, P134G, S140A, N145G, N145P, N145Q, N145T, Q146D, T151V, R159A, R159H, R159L, R159N, R159V, S161K, F166Y, N170C, N170D, P171M, A175T, A175V, Y176L, R179W, T182W, T183E, T183K, T183L, T183Q, G186I, G186L, G186P, G186T, S187E, S187T and S188E.

20. comprising, the proteolytic enzyme of claim 4, the aminoacid sequence of wherein said proteolytic enzyme is selected from least two following replacements: T10A, T10G, T10L, I11A, I11S, I11T, G12I, R14G, R14M, S15E, S15F, S15G, R16K, R16N, A22V, N24A, N24E, N24L, N24Q, N24T, N24V, G34A, T36G, T36I, T36N, T36S, A38F, A38T, G49A, G49F, S51A, G65V, L69H, L69S, Q71I, N73T, N74G, S76D, S76L, S76V, S76W, S76Y, G77T, V80A, V90I, V90P, S99N, S99V, T107K, T107R, N112S, S118A, E119R, R127F, P134D, P134E, P134H, P134L, P134R, P134V, S140A, L142V, V150L, 159F, R159K, T163I, F166Y, Q167N, N170Y, R179V, T182V, G186E, G186S and G186V.

21. the isolating serine protease variants of claim 1, wherein said variant is compared the stability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQID NO:8.

22. the variant of claim 21, wherein said variant is compared the thermostability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

23. the variant of claim 22, wherein said variant comprises and is selected from following multiple replacement: T121E/R123F/R159E, R79T/R127Q/R179Q, R16Q/R79T/R127Q, R16Q/R79T/R123L/R159Q/R179Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R123L/R159Q, R14Q/T121E, R14L/R79T, R123L/R159Q, R123L/R127Q/R159Q, G12D/S15E/R35D/R123F/R159E, G12D/S15E/R159E, G12D/S15E, G12D/R35H/T121E/R123Q, G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R123Q, G12D/R35E, G12D/R159E, G12D/R14Q/S15E/R35D, G12D/R14Q/R35H, G12D/R14Q/R159E, G12D/R14E, G12D/R127Q/R159E and G12D/R123E/R159E and R35E/R123L/R127Q/R175Q.

24. the variant of claim 21, wherein said variant is compared the LAS stability with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

25. Variant of claim 24, wherein said variant comprises multiple substitution selected from the following: T121E/R123F/R159E, S15E/T121E/R123Q, S15E/R35H/R159E, S15E/R35E/R159E, S15E/R35E/R127Q/R159E, S15E/R35E, S15E/R35D/T121E/R123Q, S15E/R35D/R123Q, S15E/R35D/R123F/R159E, S15E/R35D, S15E/R159E, S15E/R127Q, S15E/R123Q, S15E/R123E, R79T/R127Q/R179Q, R35H/R159E, R35H/R127Q/R159E, R35H/R123D/R159E, R35F/R61S/R159Q, R35F/R159Q, R35E/T121E/R123E, R35E/R159E, R35E/R127Q, R35D/R159E, R35D/R127Q/R159E, R35D/R127Q, R35D/R123Q/R159E, R16Q/R79T/R159Q/R179Q, R16Q/R79T/R127Q, R16Q/R79T/R123L/R159Q/R179Q, R16Q/R79T/R123L/R159Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R61S/R159Q/R179Q, R16Q/R61S/R123L/R159Q, R16Q/R35F/R61S/R159Q, R16Q/R35F/R159Q, R16Q/R35F/R123L/R159Q, R16Q/R35F, R16Q/R159Q/R179Q, R16Q/R159Q, R16Q/R127Q/R179Q, R16Q/R127Q/R159Q, R16Q/R123L/R159Q, R14Q/T121E, R14Q/R35E/T121E, R14Q/R35E/R159E, R14Q/R35E, R14Q/R35D/R127Q, R14Q/R35D/R123E/R159E, R14Q/R35D/R123D/R159E, R14Q/R35D, R14Q/R123Q, R14L/R79T/R127Q/R159Q, R14L/R79T, R14L/R61S/R79T/R123L, R14L/R61S/R123L, R14L/R35F/R79T/R123L/R159Q, R14L/R35F/R61S, R14L/R127Q/R159Q/R179Q, R14L/R123L/R159Q, R14I/R35E/T121E/R159E, R14I/R35E/R127Q, R14I/R35E/R123E, R14I/R35D/R159E, R14I/R35D/R127Q/R159E, R14E/S15E/R35H, R14E/R35H/R127Q, R14D/S15E/R35E/R159E, R14D/R35H/R123Q/R159E, R127Q/R159E, R127Q/R159Q, R123Q/R159E, R123Q/R127Q/R159E, R123L/R159Q, R123L/R127Q/R159Q, R123F/R159E, R123E/R127Q/R159E, R123E/R127Q, G12D/S15E/R35H/R159E, G12D/S15E/R35H/R123F/R127Q/R159E, G12D/S15E/R35E/R159E, G12D/S15E/R35D/R127Q, G12D/S15E/R35D/R123F/R159E, G12D/S15E/R35D/R123E, G12D/S15E/R35D, G12D/S15E/R159E, G12D/S15E, G12D/R35H/T121E/R123Q, G12D/R35H/R159E, G12D/R35H/R123Q/R159E, G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R159E, G12D/R35E/R123Q/R159E, G12D/R35E/R123Q, G12D/R35E, G12D/R35D/R159E, G12D/R35D/R127Q, G12D/R35D/R123Q/R159E, G12D/R35D, G12D/R159E, G12D/R14Q/S15E/R35D, G12D/R14Q/R35H, G12D/R14Q/R35E/R127Q/R159E, G12D/R14Q/R35D/R123H, G12D/R14Q/R159E, G12D/R14I/R35H, G12D/R14E, G12D/R14D/R35H/R123D/R127Q, G12D/R127Q/R159E, G12D/R123E/R159E, R127A/R159K, R14I/G65Q, R14I/G65Q/N67L/R159K, R14I/G65Q/N67L/Y75G/R127A/R159K, R14I/G65Q/R159K, R14I/G65Q/S76V/R127A/R159K, R14I/R127A, R14I/R127A/R159K, R14I/R159K, R14I/R35F, R14I/R35F/G65Q, R14I/R35F/G65Q/R127A/R159K, R14I/R35F/N67L/R127A/R159K, R14I/R35F/R127A/R159K, R14I/R35F/R159K, R14I/S76V, R14I/T36S/G65Q/R127A/R159K, R35F/R127A/R159K, R35F/S76A/R127A, N024A/G049A/A093H/S099N/R127K/A143N/R159K/I181Q, N024A/S076A/A093H/S099G/R127K/R159K, N024A/S076T/A093S/S099G/R127K/R159K, N024E/G049A/A093G/S099G/R127K/A143N/R159K/I181T, N024E/G049A/A093H / / R127K/A143N/R159K/I181Q, N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I, N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q, N024H/G049A/A093T/S099A/R127K/A143N/R159K/I181Q, N024H/S076A/A093G/S099G/R127K/R159K, N024H/S076A/A093H/S099G/R127K/R159K, N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H, N024H/S076A/A093T/S099G/R127K/R159K, N024H/S076N/A093Q/S099W/R127K/R159K, N024H/S076V/A093Q/S099G/R127K/R159K, N024L/G049A/A093H/S099A/R127K/A143N/R159K/I181Q, N024L/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N024L/S076V/A093H/S099G/R127K, N024L/S076V/A093S/S099A/R127K/R159K, N024M/G049A/A093G/S099A/R127K/A143N/R159K/I181Q, N024M/G049A/A093H/S099D/R127K/A143N/R159K/I181Q, N024M/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N024M/G049A/A093S/S099W/R127K/A143N/R159K/I181Q, N024Q/G049A/A093H/S099A/R127K/A143N/R159K/I181T, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N024Q/S076A/A093H/S099A/R127K/R159K/T039N, N024Q/S076A/A093H/S099W/R127K/R159K, N024Q/S076I/A093T/S099G/R159K, N024Q/S076T/A093S/R127K/R159K, N024S/S076A/A093G/S099G/R127K/R159K/G054A, N024S/S076A/A093H/S099T/R127K/R159K, N024S/S076A/A093S/S099W/R127K/R159K, N024S/S076A/A093T/S099W / R127K, N024S/S076T/A093Q/S099W/R127K/R159K, N024S/S076Y/A093T/S099A/R127K/R159K, N024T/G049A/A093G/S099A/R127K/A143N/R159K/I181Q, N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181Q, N024T/G049A/A093H/S099A/R127K/A143N/R159K/I181T, N024T/G049A/A093H/S099D/R127K/A143N/R159K/I181Q, N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N024T/G049A/A093T/S099A/R127K/A143N/R159K/I181T, N024T/S076N/A093Q/S099T/R127K/R159K, N024T/S076T/A093T/S099N/R127K/R159K, N024V/S076A/R127K/R159K, N024V/S076V/A093Q/S099G/R127K/R159K, N024W/A093G/S099W/R127K/R159K, N024W/G049A/A093H/S099A/R127K/A143N/R159K/I181Q, N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181K, N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N024W/S076A/A093T/S099A/R127K/R159K, N024W/S076I/A093Q/S099G/R127K/R159K, N024W/S076N/A093T/S099G/R159K, N024W/S076T/A093H/S099A/R127K/R159K, N024W/S076T/A093H/S099W/R127K/R159K, N024W/S076V/A093H/S099A/R127K/R159K, N024W/S099W/R127K/R159K, N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K, N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K, N24E/S76D/A93T/R127K/R159K, N24H/A93H/R127K/R159K, N24H/G54E/A93G/R127K/R159K, N24H/S76D/A93H/R127K/R159K, N24L/A93G/R127K/R159K, N24L/G54E/A93G/R127K/R159K, N24L/G54L/A93G/R127K/R159K, N24L/G54Q/S76A/A93H/R127K/R159K, N24L/S76T/A93G/R127K/R159K, N24L/S76T/A93H/R127K/R159K, N24M/A93G/R127K/R159K, N24M/A93H/R127K/R159K, N24M/A93S/R127K/R159K, N24M/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K, N24M/G54E/S76N/A93S/R127K/R159K, N24M/G54I/A93H/R127K/R159K/S187I, N24Q/A93G/R127K/R159K, N24Q/G54D/A93H/R127K/R159K, N24Q/G54I/A93G/R127K/R159K, N24Q/G54I/S76E/A93H/R127K/R159K, N24Q/G54Q/A93G/R127K/R159K, N24Q/G54Q/S76T/A93H/R127K/R159K, N24Q/S76A/A93G/R127K/R159K, N24T/G54D/S76V/A93G/R127K/R159K, N24T/G54E/S76V/A93H/R127K/R159K, N24T/G54I/A93G/R127K/R159K, N24T/G54N/A93H/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K, N24T/G54Q/S76V/R127K/R159K, N24T/S76I/R127K/R159K, N24T/S76L/A93G/R127K/R159K, N24W/A93G/R127K/R159K, N24W/G54D/A93H/R127K/R159K, N24W/G54I/S76A/A93H/R127K/R159K, N24W/S76A/A93H/R127K/R159K, N24W/S76E/A93G/R127K/R159K, R014I/S076A/A093G/R127K/R159K/I181T, R014I/S076A/A093H/R127K/R159K/I181K, R014I/S076A/A093H/R127K/R159K/I181Q, R014I/S076A/A093H/R127K/R159K/I181T, R014I/S076D/A093H/R127K/R 159K/I181Q, R014I/S076D/A093S/R127K/R159K/I181T, R014I/S076E/A093S/R127K/R159K/I181Q, R014I/S076E/A093T/R127K/R159K/I181K, R014I/S076I/A093S/R127K/R159K/I181Q, R014I/S076N/A093H/R127K/R159K/I181Q, R014I/S076T/A093G/R127K/R159K/I181Q, R014K/S076A/A093G/R127K/R159K/I181K, R014K/S076E/A093H/R127K/R159K/I181K, R014K/S076T/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K, R014L/S076A/A093H/R127K/R159K/I181Q, R014L/S076D/A093H/R127K/R159K/I181T, R014L/S076E/A093H/R127K/R159K/I181K, R014M/S076A/A093G/R127K/R159K/I181K, R014M/S076A/A093G/R127K/R159K/I181T, R014M/S076A/A093H/R127K/R159K/I181T, R014M/S076A/A093S/R127K/R159K/I181K, R014M/S076A/A093S/R127K/R159K/I181T, R014M/S076A/A093T/R127K/R159K/I181Q, R014M/S076D/A093S/R127K/R159K/I181T, R014M/S076E/A093G/R127K/R159K/I181T, R014M/S076E/A093H/R127K/R159K/I181T, R014M/S076E/A093S/R127K/R159K/I181T, R014M/S076N/A093G/R127K/R159K/I181K, R014M/S076N/A093G/R127K/R159K/I181T, R014M/S076N/A093H/R127K/R159K/I181Q, R014M/S076N/A093H/R127K/R159K/I181T, R014M/S076N/A093S/R127K/R159K/I181T, R014M/S076N/A093T/R127K/R159K/I181T, R014M/S076T/A093H/R127K/R159K/I181K, R014M/S076V/A093G/R127K/R159K/I181Q, R014M/S076V/A093H/R127K/R159K/I181Q, G54E/R14L, G54L/R127S, N24D/G54F/R127C, N24E/R127S, N24E/R159C, N24G/G54I/R127S, N24H/R159Y/T46I, N24I/R127V/R14V, N24T/R127Q/R179F, R127A/R159V/R179F, R127C/R14W, R127S/R159N/R123L, R14A/N24F/R159L, R14A/R127L, R14A/R127Y/R159W, R14A/R159W, R14C/S114F/R159G, R14F/R127L/R159F, R14F/R127Q/R159W, R14F/R127S/R159V, R14F/R127V/R159F, R14G/N24L/R159G, R14G/N24S/R127C, R14G/R127C/G63E, R14G/R127G, R14G/R127P, R14L/N24S/R159F, R14L/N24V/R127S/R159I, R14L/R123L, R14L/R127C/R159G, R14L/R127S, R14L/R127S/R159G, R14L/R127V, R14L/R127V/R159F, R14L/R127W/R123Y, R14L/R127Y, R14L/R127Y/R159F, R14L/R159G, R14L/R159L, R14L/R159S, R14L/R159V, R14L/R159W, R14M/N24L/R159S/R123V, R14M/R159F, R14Q/R123F, R14S/N24E/R127W, R14S/N24L/R159G, R14S/R127L/R159F, R14S/R127V, R14T/R14P/R159F, R14T/N24A, R14T/N24T/R127Q, R14T/N24T/R127Y/R159W, R14T/R127Y, R14V/N24A/R127I/R159A, R14V/N24D/R127C, R14V/N24G/P189S, R14V/N24S, R14V/N24Y/R127S/R159G, R14V/R127A, R14V/R127C/R159S, R14V/R127M/R159V, R14V/R127S/R159G, R14V/R127T/R159Y, R14V/R127V, R14V/R159F, R14V/R159V, R14V/R159W, R14W/N24T/R123E, R14W/R123L, R14W/R123V, R14W/R127Q/R159W, R14W/R159V, R159V/G49D, G012D/R035E/G065E, G012D/R035E/G065E, G012D/R035E/Q081P, G012D/R035E/R016S/A064T, G012D/R035E/R159W, G012D/R035E/R179I, G012D/R035E/S092T/I181V, R14I/N24A/A64K/R123F/R159E/D184T, R14I/A64K/R123F/R159F/D184T, R14I/A64K/R123F/R159E/D184T, R14I/N24Q/R35E/A64K/R123F/D184T, R14I/N24Q/A64K/N67S/R123F/R159F/D184T, R14I/N24A/R35E/A64K/N67S/R123F/R159E/D184T, R14I/N24A/R35E/A64K/N67S/G78D/R123F/D184T, R14I/N24A/R35D/A64K/G78D/R123F/R127K/R159E/D184T, R14I/N24A/R35D/A64K/R123F/R127K/R159F/D184T, R14I/N24T/R35D/A64K/G78D/R123F/R127Q/R159F/D184T, R14I/A64K/R123F/D184T, R14I/N24A/A64K/R123F/R159N/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R14I/N24E/R35E/A64K/G78D/R123F/R127Q/R159F/D184T, R14I/N24E/A64K/R123F/R127K/R159K/D184T, R14I/N24A/A64K/R123F/D184T, R14I/A64K/R123F/R127K/R159F/D184T, R14I/A64K/R123F/R159E/D184T, R14I/A64K/R123F/R159N/D184T, R14I/A64K/R123F/R159K/D184T, R14I/A64K/R123F/R127Y/R159E/D184T, R14I/N24A/R35E/A64K/N67A/G78D/R123F/D184T, R14I/A64K/R123F/R127Y/R159K/D184T, R14I/N24Q/A64K/R123F/R127Q/R159K/D184T, R14I/A64K/R123F/R159K/D184T, R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T, R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T, R14I/N24A/R35E/A64K/G78D/R123F/R127K/R159E/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R14I/N24A/R35D/A64K/N67A/R123F/R159F/D184T, R14I/N24E/R35E/G54D/A64K/N67L/G78D/R123F/R127K/D184T, R14I/A64K/G78D/R123F/R127Q/R159N/D184T, R14I/N24A/R35E/A64K/G78D/R123F/R159N/D184T, R14I/A64K/R123F/R127K/R159E/D184T, R14I/N24A/R35E/A64K/N67S/G78D/R123F/R127K/R159F/D184T, R14I/A64K/G78D/R123F/R159E/D184T, R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T, N24T/R35D/G78D/R159K, N24T/R35E/N67A/G78D/R127Q, N24Q/R35E, R127K/R159N, R35D/R159E, R35E/G54D/N67S/G78D/R159K, N24Q/G54D/G78D/R159N, R127K/R159E, R127Q/R159K, N24E/R35E/G54D/N67S/R127K/R159N, R35D/G78D/R159K, N67S/R159E, G54D/R127K/R159K, G78D/R127K/R159K, G78D/R127K/R159E, N24E/R35D/G78D/R127K/R159N, R35D/G78D/R127K/R159N, N24A/R35E/G78D/R159N, N24Q/R35D/N67S/R127K/R159E, N24T/R35D/G78D/R159K, N67S/G78D/R127K/R159K, N24Q/R35D/R127K/R159K, N24E/G54D/G78D/R159K, R35D/R159K, R35E/R159K, R127K/R159K, R35E/N67S/G78D/R127Q, N24E/R35D/G78D, R35D/G78D/R127K/R159E, N24E/R35E/G54D/N67S/G78D/R127K/R159K, N24T/N67S/R159E, N24D/R35D/G78D/R159F, N24Q/R35D/N67S/G78D/R127K/R159F, R35D/G78D/R127Q/R159K, G78D/R159F, N24A/N67S/R159K, G78D/R127Q/R159K, N24T/G54D/N67S/G78D/R127Y/R159E, R14I/A63K/G78D/R123F/D184T, R14I/A63K/R123F/R159E/D184T, R14I/A63K/R123F/R159F/D184T, R14I/A63K/R123F/R159K/D184T, R14I/A63K/R123F/R159N/D184T, R14I/A63K/R123K/D184T, R14I/A63K/R123Q/D184T, R14I/A63K/R123Y/D184T, R14I/A64K/G78D/T86K/T116E/R123F, R14I/A64K/T86K/T116E/R123F/R159E, R14I/A64K/T86K/T116E/R123F/R159K, R14I/A64K/T86K/T116E/R123K, R14I/A64K/T86K/T116E/R123Q, R14I/A64K/T86K/T116E/R123Y, R14I/G54D/A63K/R123F/D184T, R14I/G54D/A64K/T86K/T116E/R123F, R14I/G54D/S76N/A93H/R127K/R159K/I181Q, R14I/G54D/S76V/A93S/R127K/R159K/I181K, R14I/N24A/A63K/R123F/D184T, R14I/N24A/A64K/T86K/T116E/R123F, R14I/N24E/A63K/R123F/D184T, R14I/N24E/A64K/T86K/T116E/R123F, R14I/N24Q/A63K/R123F/D184T, R14I/N24Q/A64K/T86K/T116E/R123F, R14I/N24T/A63K/R123F/D184T, R14I/N24T/A64K/T86K/T116E/R123F, R14I/N24TS76N/A93H/R127K/R159K/I181Q, R14I/N24TS76V/A93S/R127K/R159K/I181K, R14I/N67AS76N/A93H/R127K/R159K/I181Q, R14I/N67LS76N/A93H/R127K/R159K/I181Q, R14I/N67SS76N/A93H/R127K/R159K/I181Q, R14I/R35D/A64K/T86K/T116E/R123F, R14I/R35D/S76N/A93H/R127K/R159K/I181Q, R14I/R35E/A63K/R123F/D184T, R14I/R35E/A64K/T86K/T116E/R123F, R14I/R35E/S76N/A93H/R127K/R159K/I181Q, R14I/R35E/S76V/A93S/R127K/R159K/I181K, R14I/R35K/A63K/R123F/D184T, R14I/S76N/A93H/R127K/R159E/I181Q, R14I/S76N/A93H/R127K/R159F/I181Q, R14I/S76N/A93H/R127K/R159N/I181Q, R14I/S76N/A93H/R127Q/R159K/I181Q, R14I/S76N/A93H/R127Y/R159K/I181Q, R14I/S76N/G78D/A93H/R127K/R159K/I181Q, R14I/S76V/A93S/R127K/R159F/I181K, R14I/S76V/A93S/R127K/R159N/I181K, R14I/S76V/A93S/R127Q/R159K/I181K, R14I/S76V/A93S/R127Y/R159K/I181K, R14M/G54D/S76N/A93G/R127K/R159K/I181K, R14M/N24A/S76N/A93G/R127K/R159K/I181K, R14M/N24E/S76N/A93G/R127K/R159K/I181K, R14M/N24Q/S76N/A93G/R127K/R159K/I181K, R14M/N24T/S76N/A93G/R127K/R159K/I181K, R14M/N67S/S76N/A93G/R127K/R159K/I181K, R14M/R35D/S76N/A93G/R127K/R159K/I181K, R14M/R35E/S76N/A93G/R127K/R159K/I181K, R14M/S76N/A93G/R127K/R159E/I181K, R14M/S76N/A93G/R127K/R159F/I181K, R14M/S76N/A93G/R127K/R159N/I181K, R14M/S76N/A93G/R127Q/R159K/I181K, R14M/S76N/A93G/R127Y/R159K/I181K and R14M/S76N/G78D/A93G/R127K/R159K/I181K. ...

26. the variant of claim 1, wherein said variant is compared the activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

27. the variant of claim 26, wherein said variant is compared the casein hydrolysis activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

28. the variant of claim 27, wherein said variant comprises and is selected from following multiple replacement: R14L/R79T, G12D/R35H/R159E, G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R159E, G12D/R35E/R123Q/R159E, G12D/R35E/R123Q, G12D/R35E, G12D/R35D/R159E, G12D/R35D/R123Q/R159E, G12D/R35D, G12D/R159E, G12D/R14Q/R35H, G12D/R14I/R35H, 024E/G049A/A093H/R127K/A143N/R159K/I181Q, N24M/S76V/A93H/R127K/R159K, R14I/N24E/R35D/A64K/N67A/G78D/R123F/R159K/D184T R127A/R159K, R14I/G65Q and R14I/G65Q/R159K R14I/S76V.

29. the variant of claim 26, wherein said variant is compared the Keratin sulfate hydrolytic activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

30. comprising, the variant of claim 27, wherein said variant be selected from following multiple replacement: N024E/G049A/A093H/R127K/A143N/R159K/I181Q, N024E/G049A/A093H/S099A/R127K/A143N/R159K/I181T/V090I, N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N024Q/G049A/A093S/S099N/R127K/A143N/R159K/I181Q, N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N024W/G049A/A093S/S099A/R127K/A143N/R159K/I181Q, N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K, N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K, N24E/S76D/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K, N24M/G54E/S76N/A93S/R127K/R159K, N24Q/A93G/R127K/R159K, N24Q/G54D/S76L/A93G/R127K/R159K, N24Q/G54I/S76E/A93H/R127K/R159K, N24T/G54D/S76V/A93G/R127K/R159K, N24T/G54E/S76V/A93H/R127K/R159K, N24W/G54D/A93H/R127K/R159K, N24W/S76E/A93G/R127K/R159K, R014I/S076A/A093G/R127K/R159K/I181T, R014I/S076A/A093H/R127K/R159K/I181Q, R014I/S076D/A093H/R127K/R159K/I181Q, R014I/S076D/A093H/R127K/R159K/I181T, R014I/S076D/A093S/R127K/R159K/I181T, R014I/S076E/A093S/R127K/R159K/I181Q, R014I/S076E/A093T/R127K/R159K/I181K, R014I/S076I/A093S/R127K/R159K/I181Q, R014I/S076N/A093H/R127K/R159K/I181Q, R014I/S076T/A093G/R127K/R159K/I181Q, R014I/S076V/A093H/R127K/R159K/I181Q, R014K/S076A/A093S/R127K/R159K/I181T, R014K/S076E/A093H/R127K/R159K/I181T, R014K/S076E/A093S/R127K/R159K/I181T, R014K/S076T/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K, R014L/S076A/A093H/R127K/R159K/I181Q, R014L/S076D/A093H/R127K/R159K/I181T, R014L/S076E/A093H/R127K/R159K/I181K, R014M/S076A/A093G/R127K/R159K/I181T, R014M/S076A/A093H/R127K/R159K/I181T, R014M/S076A/A093S/R127K/R159K/I181K, R014M/S076A/A093S/R127K/R159K/I181T, R014M/S076A/A093T/R127K/R159K/I181Q, R014M/S076D/A093S/R127K/R159K/I181T, R014M/S076E/A093G/R127K/R159K/I181T, R014M/S076E/A093H/R127K/R159K/I181T, R014M/S076E/A093S/R127K/R159K/I181T, R014M/S076N/A093G/R127K/R159K/I181K, R014M/S076N/A093G/R127K/R159K/I181T, R014M/S076N/A093H/R127K/R159K/I181T, R014M/S076N/A093S/R127K/R159K/I181T, R014M/S076V/A093G/R127K/R159K/I181Q, R14I/N24Q/A64K/G78D/R123F/R159K/D184T, R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R14I/N24E/A64K/R123F/R127K/R159K/D184T, R14I/N24Q/A64K/R123F/R127Q/R159K/D184T, R14I/N24Q/A64K/G78D/R123F/R127Q/R159N/D184T, R14I/N24E/A64K/N67L/G78D/R123F/R159K/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R127Q/R159K, G78D/R127K/R159K, N67S/G78D/R127K/R159K, R35D/R159K, G78D/R127Q/R159K, N24A/N67A/R159K, T36S/R127Q/R159E, S15E/T121E/R123Q, S15E/R35H/R159E, S15E/R35E, S15E/R159E, S15E/R123Q, S15E/R123E, R79T/R127Q/R179Q, R35H/R159E, R35H/R127Q/R159E, R35F/R61S/R159Q, R35F/R159Q, R35E/R159E, R35E/R127Q, R35D/R159E, R35D/R127Q, R16Q/R79T/R159Q/R179Q, R16Q/R79T/R127Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R35F/R123L/R159Q, R16Q/R159Q/R179Q, R16Q/R127Q/R159Q, R16Q/R123L/R159Q, R14Q/T121E, R14Q/R35E/T121E, R14Q/R35E/R159E, R14Q/R35E, R14Q/R35D/R127Q, R14Q/R35D, R14L/R79T/R127Q/R159Q, R14L/R61S/R79T/R123L, R14L/R35F/R61S, R14L/R127Q/R159Q/R179Q, R14L/R123L/R159Q, R14I/R35E/R127Q, R14I/R35E/R123E, R14I/R35D/R159E, R14E/R35H/R127Q, R127Q/R159E, R127Q/R159Q, R123Q/R159E, R123Q/R127Q/R159E, R123L/R159Q, R123L/R127Q/R159Q, R123F/R159E, R123E/R127Q/R159E, R123E/R127Q, G12D/S15E/R35H/R159E, G12D/S15E/R35D, G12D/S15E/R159E, G12D/S15E, G12D/R35H/T121E/R123Q, G12D/R35H/R159E, G12D/R35H/R127Q/R159E, G12D/R35H/R123Q, G12D/R35H/R123F/R159E, G12D/R35H, G12D/R35E/R159E, G12D/R35E/R123Q/R159E, G12D/R35E/R123Q, G12D/R35E, G12D/R35D/R159E, G12D/R35D/R127Q, G12D/R35D, G12D/R159E, G12D/R14Q/R35H, G12D/R14Q/R35D/R123H, G12D/R14Q/R159E, G12D/R14I/R35H, G12D/R14E, G12D/R127Q/R159E, G12D/R123E/R159E, R127A/R159K, R14I/G65Q, R14I/G65Q/R127A/R159K, R14I/G65Q/R159K, R14I/R127A, R14I/R127A/R159K, R14I/R159K, R14I/R35F, R14I/R35F/G65Q, R14I/R35F/G65Q/R127A/R159K, R14I/R35F/R159K, R14I/S76V, R14I/T36S/G65Q/R127A/R159K and R35F/R127A/R159K.

31. the variant of claim 26, wherein said variant is compared the scourability activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

32. comprising, the variant of claim 31, wherein said variant be selected from following multiple replacement: N24F/R159G ,/N24F/R159L/R123V, N24I/R127S ,/N24L/R159S, N24S/R159A, N24V/R159L, N24Y/R159F, R14A/N24K/R127S, R14A/R159W, R14L/N24Y, R14L/R159G, R14L/R159S, R14L/T109M, R14L/T39P, R14M/R159W, R14S/N24V, R14S/N24Y, R14T/N24A, R14V/N24G/P189S, R14V/R159W, R127A/R159K, R14I/G65Q, R14I/S76V, R14I/G65Q/N67L/R159K, R14I/G65Q/R159K, R14I/S76V, R35F/R159Q, R16Q/R79T, R16Q/R35F, R16Q/R159Q, R14L/R79T, R123L/R159Q, G12D/S15E, G12D/R35H, G12D/R35D, N024E/G049A/A093H//R127K/A143N/R159K/I181Q, N024H/S076A/A093S/S099A/R127K/R159K/G054H/L069H, N024H/S076A/A093T/S099G/R127K/R159K, N024L/S076V/A093S/S099A/R127K/R159K, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N024T/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N24E/G54Q/A93S/R127K/R159K, N24H/A93H/R127K/R159K, N24H/G54L/S76V/A93H/R127K/R159K, N24L/G54Q/S76A/A93H/R127K/R159K, N24M/A93G/R127K/R159K, N24M/G54E/A93H/R127K/R159K, N24M/S76V/A93H/R127K/R159K, N24Q/G54Q/S76T/A93H/R127K/R159K, N24Q/S76A/A93G/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K, N24T/S76I/R127K/R159K, N24W/G54D/A93H/R127K/R159K, N24W/S76A/A93H/R127K/R159K, N24W/S76T/A93G/R127K/R159K, N24W/S76V/A93G/R127K/R159K, N24W/S76Y/A93G/R127K/R159K, R014I/S076A/A093G/R127K/R159K/I181T, R014I/S076A/A093H/R127K/R159K/I181K, R014I/S076A/A093H/R127K/R159K/I181Q, R014I/S076A/A093H/R127K/R159K/I181T, R014I/S076D/A093S/R127K/R159K/I181T, R014I/S076E/A093T/R127K/R159K/I181K, R014I/S076N/A093H/R127K/R159K/I181Q, R014I/S076V/A093H/R127K/R159K/I181Q, R014I/S076V/A093S/R127K/R159K/I181K, R014K/S076A/A093S/R127K/R159K/I181T, R014K/S076E/A093H/R127K/R159K/I181K, R014K/S076I/A093S/R127K/R159K/I181T, R014K/S076T/A093H/R127K/R159K/I181K, R014K/S076T/A093H/R127K/R159K/I181T, R014K/S076V/A093H/R127K/R159K/I181K, R014L/S076A/A093H/R127K/R159K, R014L/S076A/A093H/R127K/R159K/I181Q, R014L/S076D/A093H/R127K/R159K/I181T, R014L/S076E/A093H/R127K/R159K/I181K, R014M/S076A/A093G/R127K/R159K/I181K, R014M/S076A/A093G/R127K/R159K/I181T, R014M/S076A/A093H/R127K/R159K/I181T, R014M/S076A/A093S/R127K/R159K/I181K, R014M/S076A/A093S/R127K/R159K/I181T, R014M/S076A/A093T/R127K/R159K/I181Q, R014M/S076I/A093H/R127K/R159K/I181T, R014M/S076I/A093S/R127K/R159K/I181T, R014M/S076N/A093G/R127K/R159K/I181K, R014M/S076N/A093G/R127K/R159K/I181T, R014M/S076N/A093H/R127K/R159K/I181T, R014M/S076N/A093S/R127K/R159K/I181T, R014M/S076T/A093H/R127K/R159K/I181K, R014M/S076V/A093G/R127K/R159K/I181Q, R014M/S076W/A093H/R127K/R159K/I181K, R014M/S076Y/A093H/R127K/R159K/I181K, R014M/S076Y/A093H/R127K/R159K/I181T, G012D/R035E/D184N, G012D/R035E/N067K, R14I/A64K/R123F/R159F/D184T, R14I/A64K/R123F/R159F/D184T, R14I/N24Q/A64K/G78D/R123F/R159K/D184T, R14I/N24Q/A64K/N67S/R123F/R159F/D184T, R14I/N24Q/A64K/N67A/R123F/R159K/D184T, R14I/N24A/A64K/N67S/G78D/R123F/R159K/D184T, R14I/A64K/R123F/D184T, R14I/N24A/A64K/R123F/R159N/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R14I/N24Q/A64K/N67A/R123F/R159K/D184T, R14I/N24E/A64K/R123F/R127K/R159K/D184T, R14I/N24A/A64K/R123F/D184T, R14I/A64K/R123F/R127K/R159F/D184T, R14I/A64K/R123F/R159N/D184T, R14I/A64K/R123F/R159K/D184T, R14I/A64K/R123F/R127Y/R159K/D184T, R14I/N24Q/A64K/R123F/R127Q/R159K/D184T, R14I/A64K/R123F/R159K/D184T, R14I/N24Q/R35D/A64K/N67S/R123F/R159K/D184T, R14I/A64K/N67S/G78D/R123F/R127K/R159K/D184T, R14I/N24Q/A64K/N67A/R123F/R127K/R159K/D184T, R127K/R159K, N24A/N67S/R159K, N24A/N67A/R159K, R14I/A63K/G78D/R123F/D184T, R14I/A63K/N67A/R123F/D184T, R14I/A63K/N67L/R123F/D184T, R14I/A63K/N67S/R123F/D184T, R14I/A63K/R123F/R159F/D184T, R14I/A63K/R123F/R159K/D184T, R14I/A63K/R123F/R159N/D184T, R14I/A63K/R123K/D184T, R14I/A63K/R123Q/D184T, R14I/A63K/R123Y/D184T, R14I/A64K/G78D/T86K/T116E/R123F, R14I/A64K/N67A/T86K/T116E/R123F, R14I/A64K/N67L/T86K/T 116E/R123F, R14I/A64K/T86K/T116E/R123F/R159K, R14I/A64K/T86K/T116E/R123K, R14I/G54D/A63K/R123F/D184T, R14I/G54D/A64K/T86K/T116E/R123F, R14I/N24A/A63K/R123F/D184T, R14I/N24A/A64K/T86K/T116E/R123F, R14I/N24A/S76V/A93S/R127K/R159K/I181K, R14I/N24E/A63K/R123F/D184T, R14I/N24E/A64K/T86K/T116E/R123F, R14I/N24Q/A63K/R123F/D184T, R14I/N24Q/A64K/T86K/T116E/R123F, R14I/N24Q/S76V/A93S/R127K/R159K/1181K, R14I/N24T/A63K/R123F/D184T, R14I/N24T/A64K/T86K/T116E/R123F, R14I/N24TS76V/A93S/R127K/R159K/I181K, R14I/N67SS76N/A93H/R127K/R159K/I181Q, R14I/R35E/A63K/R123F/D184T, R14I/R35K/A63K/R123F/D184T, R14I/S76V/A93S/R127K/R159F/I181K, R14I/S76V/A93S/R127K/R159N/I181K, R14I/S76V/A93S/R127Y/R159K/I181K, R14M/N24T/S76N/A93G/R127K/R159K/I181K, R14M/N67S/S76N/A93G/R127K/R159K/I181K, R14M/S76N/A93G/R127K/R159F/I181K and R14M/S76N/G78D/A93G/R127K/R159K/I181K.

33. the variant of claim 26, wherein said variant is compared the performance activity that washes the dishes with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

34. the variant of claim 33, wherein said variant has and is selected from following multiple replacement: R14N/R127K/R159L, R14I/A64K/T86K/N112E/R123F/D184T, G12D/R35E/G63R/R79K/T109M, R14L, G12D/R35E and R14M/S76D/A93H/R127K/R159K/I181K.

35. the variant of claim 26, wherein said variant is compared the soil release activity with raising with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQ ID NO:8.

36. comprising, the variant of claim 35, wherein said variant be selected from following multiple replacement: G54E/R14L, N24D/R127Y/R159V, N24E/R127S, N24E/R159C, N24F/R159G, N24F/R159G/G54E, N24F/R159L/R123V, N24G/R127Y, N24H/R159Y/T46I, N24I/R127S, N24I/R127V/R14/N24L/R159S, N24S/R159A, N24V/R127M/R159V, N24V/R127S/R159H, N24V/R159L, N24Y/G54A, N24Y/R127L, N24Y/R127S, N24Y/R127V, N24Y/R159F, R127A/R159F, R127H/R159Q, R127H/R159T/S185F, R127M/R159V, R127S/R159G, R127S/R159L, R127T/R159F, R127V/R159G, R127Y/R159L, R14A/N24K/R127S, R14A/R127Y/R159W, R14G/N24S/R127C, R14G/R127G, R14L/N24D, R14L/N24Y, R14L/R123L, R14L/R127S, R14L/R127V, R14L/R127Y, R14L/R159G, R14L/R159S, R14L/T39P, R14M/N24L/R159S/R123V, R14M/R159F, R14M/R159W, R14Q/R123F, R14S/N24E/R127W, R14S/N24V, R14S/N24Y, R14T/N24T/R127Q, R14T/R127Y, R14V/N24D/R127C, R14V/N24G/P189S, R14V/N24L/R127F, R14V/R127A, R14V/R159F, R14V/R159W, R14W/N24A, R14W/R123L, R14W/R123V, R14W/R159V, R159V/G49D, R159V/R123G, N024E/G049A/A093H/R127K/A143N/R159K/I181Q, N024E/G049A/A093S/S099D/R127K/A143N/R159K/I181Q, N024Q/G049A/A093S/S099A/R127K/A143N/R159K/I181T, N24A/G54E/S76D/A93G/R127K/R159K, N24E/A93G/R127K/R159K, N24E/G54L/S76E/A93G/R127K/R159K, N24E/G54Q/A93S/R127K/R159K, N24E/S76D/A93T/R127K/R159K, N24H/G54E/A93G/R127K/R159K, N24L/S76T/A93G/R127K/R159K, N24M/A93S/R127K/R159K, N24M/A93T/R127K/R159K, N24M/G54E/A93H/R127K/R159K, N24M/G54E/S76N/A93S/R127K/R159K, N24M/S76V/A93H/R127K/R159K, N24Q/A93G/R127K/R159K, N24Q/G54D/S76L/A93G/R127K/R159K, N24Q/G54I/S76T/A93G/R127K/R159K, N24Q/G54Q/A93G/R127K/R159K, N24Q/S76A/A93G/R127K/R159K, N24T/G54Q/S76N/A93G/R127K/R159K, N24T/G54Q/S76V/R127K/R159K, N24T/S76I/R127K/R159K, N24W/A93G/R127K/R159K, N24W/G54D/A93H/R127K/R159K, R014I/S076A/A093G/R127K/R159K/I181T, R014I/S076A/A093H/R127K/R159K/I181Q, R014I/S076D/A093H/R127K/R159K/I181Q, R014I/S076D/A093H/R127K/R159K/I181T, R014I/S076D/A093S/R127K/R159K/I181T, R014I/S076E/A093S/R127K/R159K/I181Q, R014I/S076E/A093T/R127K/R159K/I181K, R014I/S076N/A093H/R127K/R159K/I181Q, R014L/S076A/A093H/R127K/R159K, R014L/S076A/A093H/R127K/R159K/I181Q, R014L/S076D/A093H/R127K/R159K/I181T, R014M/S076A/A093G/R127K/R159K/I181T, R014M/S076A/A093S/R127K/R159K/I181K, R014M/S076A/A093S/R127K/R159K/I181T, R014M/S076A/A093T/R127K/R159K/I181Q, R014M/S076D/A093S/R127K/R159K/I181T, R014M/S076E/A093H/R127K/R159K/I181T, R014M/S076E/A093S/R127K/R159K/I181T, R014M/S076N/A093G/R127K/R159K/I181K, R014M/S076N/A093G/R127K/R159K/I181T, R014M/S076N/A093H/R127K/R159K/I181T, R014M/S076N/A093S/R127K/R159K/I181T, T36S/R127Q/R159E, S15E/R35E, S15E/R35D, S15E/R159E, S15E/R127Q, S15E/R123Q, S15E/R123E, R79T/R127Q/R179Q, R35H/R159E, R35F/R61S/R159Q, R35F/R159Q, R35D/R127Q, R16Q/R79T/R159Q/R179Q, R16Q/R79T/R123L, R16Q/R79T, R16Q/R35F, R16Q/R159Q/R179Q, R16Q/R159Q, R14Q/T121E, R14Q/R35E, R14Q/R35D, R14Q/R123Q, R14L/R79T, R127Q/R159E, R127Q/R159Q, R123Q/R159E, R123L/R159Q, R123F/R159E, G12D/S15E, G12D/R35H/R159E, G12D/R35H/R123Q, G12D/R35H, G12D/R35E, G12D/R35D, G12D/R159E, G12D/R14Q/R35H, G12D/R14I/R35H, G65Q/R127A/R159K, R127A/R159K, R14I/G65Q, R14I/G65Q/N67L/R159K, R14I/G65Q/R127A, R14I/G65Q/R127A/R159K, R14I/G65Q/R159K, R14I/R127A, R14I/R127A/R159K, R14I/R159K, R14I/R35F, R14I/R35F/G65Q, R14I/R35F/R159K, R14I/S76V and R35F/R127A/R159K.

37. the isolating serine protease variants of claim 1, wherein said variant are compared the surface property of display change with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQID NO:8.

38. being included in, the proteolytic enzyme of claim 37, wherein said variant is selected from following locational at least two replacements: 1,2,4,7,8,10,11,12,13,14,15,16,22,24,25,32,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,57,59,61,62,63,64,65,66,67,68,69,71,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,95,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,123,124,126,127,128,130,131,132,133,134,135,137,143,144,145,146,147,148,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,173,174,175,176,177,178,179,180,181,182,183 and 184, carry out on the position of wherein said at least two replacements position in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ ID NO:8.

39. comparing with the wild-type Cellulomonas 69B4 proteolytic enzyme that comprises aminoacid sequence shown in the SEQID NO:8, the isolating serine protease variants of claim 1, wherein said variant have at least a improved characteristic.

40. the proteolytic enzyme of claim 39, wherein said at least a improved characteristic is selected from acid acceptance, thermostability, casein hydrolysis, Keratin sulfate hydrolysis, clean-up performance and LAS stability.

41. expression vector, it comprises the polynucleotide sequence of the described isolating serine protease variants of coding claim 1.

42. host cell, it comprises the described expression vector of claim 41.

43. the host cell of claim 42, it is mould that wherein said host is selected from genus bacillus, streptomycete, aspergillus and wood.

44. serine protease variants, it is produced by the described host cell of claim 43.

45. composition, it comprises at least a portion of the described isolating serine protease variants of claim 1.

46. polynucleotide sequence, the described isolating serine protease variants of its coding claim 1.

47. expression vector, it comprises the polynucleotide sequence of claim 46.

48. host cell, it comprises the described expression vector of claim 47.

49. the host cell of claim 48, it is mould that wherein said host is selected from genus bacillus, streptomycete, aspergillus and wood.

50. serine protease variants, it is produced by the described host cell of claim 49.

51. cleaning compositions, it comprises the described serine protease variants of at least a claim 1.

52. cleaning compositions, it comprises the variant serine protease of at least a claim 1, and the Serine Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence has the immunology cross reactivity shown in wherein said serine protease variants and the described SEQ of the comprising ID NO:8.

53. the cleaning compositions of claim 51, wherein said replacement are in being equivalent to comprise the Cellulomonas 69B4 proteolytic enzyme of aminoacid sequence shown in the SEQ IDNO:8 1,2,3,4,7,8,9,10,11,12,13,14,15,16,18,19,22,24,25,26,27,28,29,30,31,32,33,34,35,36,38,39,40,41,42,43,44,45,46,47,48,49,51,52,54,55,56,57,59,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,83,84,85,86,87,88,89,90,91,92,93,96,99,100,101,103,104,105,107,109,110,111,112,113,114,115,116,117,118,119,121,123,124,125,126,127,128,129,130,132,133,134,135,136,137,140,141,142,143,144,145,146,147,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,170,171,172,175,176,177,179,180,181, carry out on 182,183,184,185,186,187,188 and 189 bit positions.

54. the cleaning compositions of claim 53, it also comprises one or more and is selected from following other enzymes or enzyme derivative: proteolytic enzyme, amylase, lipase, mannase, polygalacturonase, at, oxydo-reductase, hemicellulase and cellulase.

55. the cleaning compositions of claim 51, it also comprises at least a stablizer.

56. the composition of claim 55, wherein said stablizer is selected from borax, glycerine and competitive inhibitor.

57. the composition of claim 56, wherein said competitive inhibitor make described serine protease stable to aniorfic surfactant.

58. the composition of claim 55, wherein said serine protease are the stable variant of self-dissolving.

59. cleaning compositions, it comprises the described serine protease of claim 1 of at least 0.0001 weight percent and supplementary component randomly.

60. the cleaning compositions of claim 59, wherein said composition comprise the about 0.001 described serine protease to about 0.5 weight percent.

61. the cleaning compositions of claim 59, wherein said composition comprise the about 0.01 described serine protease to about 0.1 weight percent.

62. the cleaning compositions of claim 61, wherein said composition comprises supplementary component.

Think that described composition provides about 3 to about 5 clean pH 63. the cleaning compositions of claim 59, described composition comprise the pH regulator agent of q.s, described composition does not contain the material of hydrolysis under about 3 to about 5 pH substantially.

64. the cleaning compositions of claim 63, the material of wherein said hydrolysis comprises surfactant materials.

65. the cleaning compositions of claim 64, wherein said surfactant materials comprise the alkylsurfuric acid natrium surfactant that comprises ethylene oxide moiety.

66. the cleaning compositions of claim 51, wherein said cleaning compositions is selected from liquid, powder, particulate state and tablet composition.

67. the cleaning compositions of claim 51, wherein said composition also comprise the hydrogen peroxide source.

68. the cleaning compositions of claim 67, wherein said hydrogen peroxide source comprises at least a persalt, and wherein said persalt is alkali metal perborate, alkali metal percarbonate, basic metal superphosphate, alkali metal persulphate or its mixture.

69. the cleaning compositions of claim 68, wherein said composition also comprise bleaching catalyst, bleach activator and/or its mixture.

70. cleaning method said method comprising the steps of:

A) will comprise the surface of fabric and/or the cleaning compositions of article contact claim 51; With

B) randomly the washing and/or described surface of rinsing or material.

71. cleaning method said method comprising the steps of:

A) will comprise the surface of fabric and/or the cleaning compositions of article contact claim 59; With

B) randomly the washing and/or described surface of rinsing or material.

72. cleaning method said method comprising the steps of:

A) will comprise the surface of fabric and/or the cleaning compositions of article contact claim 63; With

B) randomly the washing and/or described surface of rinsing or material.

73. cleaning method said method comprising the steps of:

A) will comprise the surface of fabric and/or the cleaning compositions of article contact claim 64; With

B) randomly the washing and/or described surface of rinsing or material.

74. cleaning method said method comprising the steps of:

A) will comprise the surface of fabric and/or the cleaning compositions of article contact claim 67; With

B) randomly the washing and/or described surface of rinsing or material.

75. animal-feed, it comprises the serine protease variants of claim 1.

76. textiles or leather processing composition, it comprises the serine protease variants of claim 1.