EP0758373A1

EP0758373A1 - Fabric cleaning compositions containing subtilisin bpn' variants

Info

Publication number: EP0758373A1
Application number: EP95916426A
Authority: EP
Inventors: Philip Frederick Brode, Iii; Bobby Lee Barnett; Donn Nelton Rubingh; Chanchal Kamur Ghosh
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1994-05-02
Filing date: 1995-04-17
Publication date: 1997-02-19
Also published as: BR9507593A; NO964590D0; AU2293195A; CA2189428A1; ZA952220B; CN1151757A; PE49595A1; MX9605359A; WO1995029979A1; NO964590L; FI964411A; FI964411A0; PE12396A1; JPH09512433A

Abstract

The present invention relates to fabric cleaning compositions comprising subtilisin BPN' variants, wherein the BPN' variant comprises one or more amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions, whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'.

Description

FABRIC CLEANING COMPOSITIONS CONTAINING SUBTILISIN BPN' VARIANTS

TECHNICAL FIELD The present invention relates to fabric cleaning compositions comprising protease enzymes which are subtilisin variants.

BACKGROUND

Enzymes make up the largest class of naturally occurring proteins.

Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction. One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.

Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.

The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.

Despite the variety of approaches described in the art, there is a continuing need for compositions comprising effective variants of proteases useful for cleaning fabric surfaces. Objects of the Present Invention It is an object of the present invention to provide fabric cleaning compositions comprising subtilisin enzyme variants.

SUMMARY The present invention relates to compositions comprising subtilisin

BPN' variants for cleaning fabric surfaces. The BPN' variants useful in these compositions comprise at least one, two or three amino acid positions having a different amino acid than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions, whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'.

DESCRIPTION I. Subtilisin Variants Useful In Fabric Cleaning Compositions

This invention relates to fabric cleaning compositions comprising a subtilisin enzyme, in particular BPN', that has been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, "BPN¹ variants") useful in the compositions of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. Certain of these BPN' variants are described in co-pending application U.S.S.N. 08/121 ,437, filed September 15, 1993 by Brode et al.

The subtilisin enzymes useful in the compositions of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.

The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case. (Brode, P.F. Ill and D. S. Rauch, LANGMUIR, "Subtilisin BPN': Activity on an Immobilized Substrate", Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface," in POLYMER SOLUTIONS, BLENDS AND INTERFACES, Ed. by I. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)). Surprisingly, when seeking to apply this principle in the search for variant proteases which give better fabric cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).

While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface. The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils. In BPN', the amino acids from position 199 to position 220 form a large exterior loop on the enzyme molecule. It has been discovered that this loop plays a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in this loop have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that this loop is important to the adsorption of the BPN' molecule for at least two reasons. First, the amino acids which comprise this exterior loop can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of this loop to the active-site and binding pocket of the BPN' molecule gives it a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).

As used herein, "variant" means an enzyme having an amino acid sequence which differs from that of wild-type. As used herein, "mutant BPN' gene" means a gene coding for a BPN' variant.

As used herein, "wild-type subtilisin BPN'" refers to a subtilisin enzyme represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPN' is further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell and E. Y. Chen, NUCLEIC ACIDS RESEARCH, Vol. II, 7911-7925 (1983), incorporated herein by reference. As used herein, the term "wild-type amino acid sequence" encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 199-220.

As used herein, "more hydrophilic amino acid" refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1 ) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T.P., and Woods, K.R., "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981 , incorporated herein by reference).

Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9). The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).

Therefore, for example, the statement "substitute Gin with an equally or more hydrophilic amino acid which is neutral or has a negative charge" means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin. Likewise, the statement "substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge" means Pro would be substituted with Gin, Asn, Ser, Glu or Asp. A. Variants comprising at least one amino acid substitution

In one embodiment of the present invention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence at one or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 218, 219 or 220 is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'. Preferably, the positions having a substituted amino acid are 199, 200, 201 , 202, 205, 207, 208, 209, 210, 211 , 212 or 215; more preferably, 200, 201 , 202, 205 or 207.

Preferably, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 201 is Gly, Gin,

Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 203 is Met, Cys, His, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 204 is Glu.

Preferably, the substituting amino acid for position 205 is Leu, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 206 Pro, Asn or Ser.

Preferably, the substituting amino acid for position 207 is Asp or Glu.

Preferably, the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 210 is Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 212 is Gin, Ser,

Asp or Glu. Preferably, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu. Preferably, the substituting amino acid for position 215 is Thr, Pro,

Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 218 is Glu. Preferably, the substituting amino acid for position 219 is Pro, Gin,

Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Asp or Glu.

More preferably, the substituting amino acid for any of positions 199, 200, 201 , 202, 203, 205, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216,

219 and 220 is, with reference to Table 1 , is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'.

More preferably still, the substituting amino acid for any of positions 199, 200, 201 , 202, 203, 205, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 219 and 220 is Asp, or Glu; and the substituting amino acid for positions 204 or 218 is Glu. B. Variants comprising at least two amino acid substitutions

In another embodiment of the present invention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence at two or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 or 220 is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'. Preferably, the positions having a substituting amino acid are 199, 200, 201, 202, 205, 207, 208, 209, 210, 211 , 212, or 215; more preferably, positions 200, 201 , 202, 205 or 207.

Preferably, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 200 is His, Thr,

Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 204 is Asp or Glu.

Preferably, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu. Preferably, the substituting amino acid for position 207 is Asp or Glu.

Preferably, the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 210 is Ala, Gly,

Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 212 is Gin, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly. Gin, Asn, Ser, or Asp.

Preferably, the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 215 is Thr, Pro,

Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 218 is Gin, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 220 is Pro, Gly,

Gin, Asn, Ser, Asp or Glu.

More preferably, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 or 220 is, with reference to Table 1 , is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'. More preferably still, the substituting amino acid for any of positions

199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 214, 215, 216, 218, 219 or 220 is Asp and Glu; for position 217 is Leu, Asp, or Glu; and for position 213 is Asp.

C. Variants comprising at least three amino acid substitutions In another embodiment of the present invention, the BPN' variant comprises wild-type amino acid sequence wherein the wild-type amino acid sequence of three or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220, is substituted; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild- type subtilisin BPN'. Preferably, the positions having a substituting amino acid are 199, 200, 201 , 202, 205, 207, 208, 209, 210, 211 , 212, or 215; more preferably positions 200, 201 , 202, 205 or 207.

Preferably, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 202 is Pro, Gin,

Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 203 Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 204 is selected from the group consisting of Asp or Glu.

Preferably, the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 209 is lie, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 210 is Ala, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 211 is Ala, Pro,

Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 215 is Thr, Pro, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. Preferably, the substituting amino acid for position 218 is Gin, Ser,

Asp or Glu.

Preferably, the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu.

Preferably, the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Ser Asp or Glu.

More preferably, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220 is, with reference to Table 1, is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin BPN'.

More preferably still, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 218, 219 or 220 is Asp or Glu; for position 217 is Leu, Asp, or Glu; and for position 213 is Asp. D. Preparation of enzyme variants

Example 1

Mutant BPN' Genes

A phagemid (pSS-5) containing the wild type subtilisin BPN' gene (Mitchinson, C. and J. A. Wells, (1989), "Protein Engineering of Disulfide

Bonds in Subtilisin BPN', BIOCHEMISTRY, Vol. 28, pp. 4807-4815) is transformed into Escheήchia coli ung-strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel, T.A., J.D. Roberts and R.A. Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOLOGY, Vol. 154, pp. 367-382, (1987); as modified by Yuckenberg, P.D., F. Witney, J. Geisselsoder and J. McClary, "Site-directed in vitro mutagenesis using uracil-containing DNA and phagemid vectors", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed. M.J. McPherson, pp. 27-48, (1991 ); both of which are incorporated herein by reference). A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M.J., and M. Smith, "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS RESEARCH, Vol. 10, pp. 6487-6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al., 1991, above). Oligonucleotides are made using an Applied Biosystem Inc. 380B DNA synthesizer. Mutagenesis reaction products are transformed into Escheήchia coli strain MM294 (American Type Culture Collection E. Coli. 33625). All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M. Y., E. Ferrari and D. J. Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtillis and the Use of the Cloned Gene to Create an In Wfro-derived Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol. 160, pp. 15-21 ). For some of the mutants a modified pSS-5 with a frameshift-stop codon mutation at amino acid 217 is used to produce the uracil template. Oligonucleotides are designed to restore the proper reading frame at position 217 and also encoded for random substitutions at positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.

Example 2 Fermentation The Bacillus subtilis cells (BE2036) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B. Braun Biotech, Inc., Allentown, Pennsylvania) in a total volume of 10 liters. The fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION: A PRACTICAL APPROACH, Ed. B. McNeil and L. M. Harvey, 1990). The broth is kept at a constant pH of 7.0 during the fermentation run. Chloramphenical is added for antibiotic selection of mutagenized plasmid. The cells are grown overnight at 37°C to an AQOO °f about 60 and harvested. Example 3

Purification The fermentation broth is taken through the following steps to obtain pure enzyme. The broth is cleared of Bacillus subtilis cells by centrifugation, and clarified by removing fine particulates with a 100K cutoff membrane. This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5.5 using 0.025M MES buffer (2-(Λ/-morpholino)ethanesulfonic acid). The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI or a propylene glycol gradient (See Scopes, R. K., PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Springer-Verlag, New York (1984), incorporated herein by reference).

The pNA assay (DelMar, E.G., C. Largman, J.W. Brodrick and M.C. Geokas, ANAL BIOCHEM., Vol. 99, pp. 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein

SUBSTITUTE SHEET^"(RULE 26) concentration. The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution.

To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type ll-T: turkey egg white purchased from Sigma Chemical Company (St. Louis, Missouri). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA.

In preparation for use, the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCl2 and pH adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C.

E. Characterization of enzyme variants

Example 4 Model Surface Preparation Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, California). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3- (dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 70°C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P.F. Ill, and D.S. Rauch, "Subtilisin BPN': Activity on an Immobilized Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992), incorporated herein by reference.

The CPG surface will have 62,000 ± 7,000 pNA molecules/μm². The surface area will remain unchanged from the value of 50.0π.2/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).

Example 5 Surface Hydrolysis Assay

Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment. A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed. The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e.g., the first 20 minutes - and every 10 minutes towards the end of the experiment). The CPG:sAAPF-pNA is allowed to settle and the solution is sampled. Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode er a/., 1992, above.

All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment. Therefore, enzyme adsorption can be determined by measuring solution depletion. The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed. The amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm. The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask. This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme.

Example 6 Soluble Substrate Kinetic Analysis The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer. The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 μM sAAPF-pNA for each kinetic determination. An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUS™ spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts). Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v₀. The v₀ and s₀ data are plotted in the standard inverse fashion to give KM and k_cat- F. Example BPN' variants

BPN' variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Table 2, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third. TABLE 2 Example BPN' Variants _:

-Single Mutation—

Ala216Glu

Ala216Asp

Ala216Gly Val203Glu

-Double Mutation-

Ile205Leu + Ala216Glu

Ile205Leu + Ala216Asp Pro210Ala + Gly215Thr

Tyr214Phe + Tyr217Asn Gln206Glu + Ala216Glu Ala216Glu + Try217Leu Gln206Glu + Tyr217Leu

-Triple Mutation- Gln206Glu + Ala216Glu + Tyr217Leu Gln206Pro + Gly211Ala + Ala216Glu -Quadruple Mutation-

Val203Glu + Gln206Glu + Ala216Glu + Tyr217Leu Val203Glu + Pro210Ala + Ala216Glu + Tyr217Leu

-Quintuple Mutation- Val203Glu + Gln206Glu + Gly215Thr + Ala216Glu + Tyr217Leu Val203Glu + Pro210Ala + Gly215Thr + Ala216Glu + Tyr217Leu

II. Fabric Cleaning Composition Materials

The fabric cleaning compositions of the present invention also comprise, in addition to the BPN' variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme. The term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid; granule; bar), which materials are also compatible with the BPN' variant used in the composition. The specific selection of cleaning composition materials are readily made by considering the fabric to be cleaned, and the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term "compatible", as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the BPN' variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.

As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form.

As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular) composition is required, and the like. Preferably the cleaning compositions of the present invention comprise from about 0.0001% to about 10% of one or more enzyme variants, more preferably from about 0.001 % to about 1%, more preferably still from about 0.01% to about 0.1%. Several examples of various cleaning compositions of the present invention are discussed in further detail below. All parts, percentages and ratios used herein are by weight unless otherwise specified.

The enzyme variants of the present invention can be used with various conventional ingredients to provide fully-formulated fabric laundering compositions. Such compositions can be in the form of liquids, granules and the like. Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants.

The fabric cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.

Nonlimiting examples of surfactants useful herein include the conventional C-| 1-C<|8 alkyl benzene sulfonates and primary and random alkyl sulfates, the C10- 18 secondary (2,3) alkyl sulfates of the formulas CH₃(CH₂)x(CHOSO3)-M⁺)CH₃ and CH₃(CH₂)y(CHOS0₃-M⁺) CH₂CH₃ wherein x and (y+1) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the C-|o^_ C-|8 alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C-|0" i8 alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C-JO- C-|8 a'kyl polyglycosides, and their corresponding sulfated polyglycosides, C-J2- -18 alpha-sulfonated fatty acid esters, C12- 18 ^a' ^ar|d ^a' phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C12-C18 betaines and sulfobetaines ("sultaines"), C10- 18 amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C18 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al., issued March 16, 1993, incorporated herein by reference.

A wide variety of other ingredients useful in fabric cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C1 -C 6 alkolamides can be incoφorated into the compositions, typically at about 1 % to about 10% levels. The C-10- 1 monoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2, MgSO- , and the like, can be added at levels of, typically, from about 0.1% to about 2%, to provide additionally sudsing. The liquid fabric cleaning compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1 ,3-propanediol, ethylene glycol, glycerine, and 1 ,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.

The fabric cleaning compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

When formulating the fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies.

Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001 % to about 1% by weight. Various fabric care enzymes are well-known in the laundry detergent art.

Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.

Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials. Enzyme stabilizers may also be used in the cleaning compositions of the present invention. Such enzyme stabilizers include propylene glycol (preferably from about 1 % to about 10%), sodium formate (preferably from about 0.1 % to about 1 %) and calcium formate (preferably from about 0.1 % to about 1 %). a. Granular fabric cleaning compositions

The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1% by weight of active enzyme of the composition. In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.

The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 7-10 Granular Fabric Cleaning Composition

Example No. Component 8 9 10

Ala216Glu 0.10 0.20 0.03 0.05

Gln206Glu + Tyr217Leu - - 0.02 0.05

C-|₃ linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00

Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates)

Sodium carbonate 23.00 23.00 23.00 23.00

Sodium silicate 14.00 14.00 14.00 14.00

Zeolite 8.20 8.20 8.20 8.20

Chelant (diethylaenetriamine- 0.40 0.40 0.40 0.40 pentaacetic acid)

Sodium sulfate 5.50 5.50 5.50 5.50

Water balance to 100%

In Examples 7-8, the BPN' variants recited in Table 2, among others, are substituted for Ala216Glu, with substantially similar results.

In Examples 9-10, any combination of the BPN' variants recited in Table 2, among others, are substituted for Ala216Glu and Gln206Glu + Tyr217Leu, with substantially similar results. Examples 11-14 Granular Fabric Cleaning Composition

Example No.

Component 11 12 13 14

Gln206Glu + Ala216Glu + Tyr217Leu 0.10 0.20 0.03 0.05

Pro210Ala + Gly215Thr - - 0.02 0.05

C12 alkyl benzene sulfonate 12.00 12.00 12.00 12.00

Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00

2-butyl octanoic acid 4.00 4.00 4.00 4.00

C12-C14 secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00

Na salt

Sodium citrate 5.00 5.00 5.00 5.00

Optical brightener 0.10 0.10 0.10 0.10

Sodium sulfate 17.00 17.00 17.00 17.00

Water and minors balance to 100%

In Examples 11-12, the BPN' variants recited in Table 2, among others, are substituted for Gln206Glu + Ala216Glu + Tyr217Leu, with substantially similar results.

In Examples 13-14, any combination of the BPN' variants recited in Table 2, among others, are substituted for Gln206Glu + Ala216Glu + Tyr217Leu and Pro210Ala + Gly215Thr, with substantially similar results.

Examples 15 and 16 Granular Fabric Cleaning Compositions Components Example No.

15 16

Linear alkyl benzene sulphonate 11.4 10.70

Tallow alkyl sulphate 1.80 2.40

Cl 4-15 alkyl sulphate 3.00 3.10

Ci4_<i5 alcohol 7 times ethoxylated 4.00 4.00

Tallow alcohol 11 times ethoxylated 1.80 1.80

Dispersant 0.07 0.1

Silicone fluid 0.80 0.80

Trisodium citrate 14.00 15.00

Citric acid 3.00 2.50

Zeolite 32.50 32.10

Maleic acid acrylic acid copolymer 5.00 5.00

Diethylene triamine penta methylene 1.00 0.20 phosphonic acid Ala216Glu + Tyr217Leu 0.30 0.30

Lipase 0.36 0.40

Amylase 0.30 0.30

Sodium silicate 2.00 2.50

Sodium sulphate 3.50 5.20

Polyvinyl pyrrolidone 0.30 0.50

Perborate 0.5 1

Phenol sulphonate 0.1 0.2

Peroxidase 0.1 0.1

Minors Up to 100 Up to 100 Examples 17 and 18

Granular Fabric Cleaning Compositions

Example No.

Components 1Z 18

Sodium linear C12 alkyl I benzene-sulfonate 6.5 8.0

Sodium sulfate 15.0 18.0

Zeolite A 26.0 22.0

Sodium nitrilotriacetate 5.0 5.0

Polyvinyl pyrrolidone 0.5 0.7

Tetraacetylethylene diamine 3.0 3.0

Boric acid 4.0 -

Perborate 0.5 1

Phenol sulphonate 0.1 0.2

He205Leu + Ala216Glu 0.4 0.4

Fillers (e.g., silicates; carbonates; perfumes; Up to 100 Up to 100 water)

Example 19

Compact Granular Fabric Cleaning Composition

Components Weight %

Alkyl Sulphate 8.0

Alkyl Ethoxy Sulphate 2.0 Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6.0

Polyhydroxy fatty acid amide 2.5

Zeolite 17.0

Layered silicate/citrate 16.0

Carbonate 7.0

Maleic acid acrylic acid copolymer 5.0

Soil release polymer 0.4 21

Carboxymethyl cellulose 0.4

Poly (4-vinylpyridine) -N-oxide 0.1

Copolymer of vinylimidazole and vinylpyrrolidone 0.1

PEG2000 0.2

Val203Glu + Gln206Glu + Ala216Glu + Tyr217Leu 0.5

Lipase 0.2

Cellulase 0.2

Tetracetylethylene diamine 6.0

Percarbonate 22.0

Ethylene diamine disuccinic acid 0.3

Suds suppressor 3.5

Disodium-4,4'-bis (2-morpholino -4-anilino-s- ^■triazin-6- 0.25 ylamino) stilbene-2,2'-disulphonate

Disodium-4,4'-bis (2-sulfostyril) biphenyl 0.05

Water, Perfume and Minors Up to 100

Example 20

Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulphonate 7.6

C16-C18 alkyl sulfate 1.3

C- 14-15 alcohol 7 times ethoxylated 4.0

Coco-alkyl-dimethyl hydroxyethyl ammonium chloride 1.4

Dispersant 0.07

Silicone fluid 0.8

Trisodium citrate 5.0

Zeolite 4A 15.0

Maleic acid acrylic acid copolymer 4.0

Diethylene triamine penta methylene phosphonic acid 0.4

Perborate 15.0

Tetraacetylethylene diamine 5.0

Smectite clay 10.0

Poly (oxy ethylene) (MW 300,000) 0.3

Tyr214Phe + Tyr217Asn 0.4

Lipase 0.2

Amylase 0.3

Cellulase 0.2

Sodium silicate 3.0

Sodium carbonate 10.0 Carboxymethyl cellulose 0.2

Brighteners 0.2

Water, perfume and minors Up to 100

Example 21 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulfonate 6.92

Tallow alkyl sulfate 2.05

C14-15 alcohol 7 times ethoxylated 4.4

C 12-15 alkyl ethoxy sulfate - 3 times ethoxylated 0.16

Zeolite 20.2

Citrate 5.5

Carbonate 15.4

Silicate 3.0

Maleic acid acrylic acid copolymer 4.0

Carboxymethyl cellulase 0.31

Soil release polymer 0.30

Val203Glu + Pro210Ala + Gly215Thr + Ala216Glu + 0.2 Tyr217Leu

Lipase 0.36

Cellulase 0.13

Perborate tetrahydrate 11.64

Perborate monohydrate 8.7

Tetraacetylethylene diamine 5.0

Diethylene tramine penta methyl phosphonic acid 0.38

Magnesium sulfate 0.40

Brightener 0.19

Perfume, silicone, suds suppressors 0.85

Minors Up to 100 b. Liguid fabric cleaning compositions

Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition. Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water. The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 22-26 Liquid Fabric Cleaning Compositions Example No.

Component 22 23 24 25 26

Gln206Glu + Ala216Glu + Tyr217Leu 0.05 0.03 0.30 0.03 0.10 Pro210Ala + Gly215Thr — — — 0.01 0.20 C-I2- C<|4 alkyl sulfate, Na 20.00 20.00 20.00 20.00 20.00 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C^<ιo alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 Monethanolamine 2.50 2.50 2.50 2.50 2.50 Water/propylene olvcol/ethanol (10 000::11 ::11 )) balance to 100%

In Examples 22-24 the BPN' variants recited in Table 2, among others, are substituted for Gln206Glu + Ala216Glu + Tyr217Leu, with substantially similar results.

In Examples 25-26, any combination of the BPN' variants recited in Table 2, among others, are substituted for Gln206Glu + Ala216Glu + Tyr217Leu and Pro210Ala + Gly215Thr, with substantially similar results.

Examples 27-28 Liguid Fabric Cleaning Compositions

Example No.

Component 27 28

C 12-14 alkenyl succinic acid 3.0 8.0

Citric acid monohydrate 10.0 15.0

Sodium C 2-15 alkyl sulphate 8.0 8.0 Sodium sulfate of C 2-15 alcohol 2 times ethoxylated - 3.0

C 2-15 alcohol 7 times ethoxylated - 8.0

C 12-15 alcohol 5 times ethoxylated 8.0 -

Diethylene triamine penta (methylene phosphonic acid) 0.2 -

Oleic acid 1.8 - Ethanol 4.0 4.0

Propanediol 2.0 2.0

Ala216Glu +Tyr217Leu 0.2 0.2

Polyvinyl pyrrolidone 1.0 2.0 Suds suppressor 0.15 0.15 NaOH up to pH 7.5 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.4 0.1 Waters and minors up to 100 parts

In each of Examples 27 and 28 herein, the BPN' variants recited in Table 2, among others, are substituted for Ala216Glu +Tyr217Leu, with substantially similar results.

Examples 29-31 Liguid Fabric Cleaning Compositions

Example No.

Component 29 30 31

Citric Acid 7.10 3.00 3.00

Fatty Acid - 2.00 2.00

Ethanol 1.93 3.20 3.20

Boric Acid 2.22 3.50 3.50

Monoethanolamine 0.71 1.09 1.09

1,2 Propanediol 7.89 8.00 8.00

NaCumene Sulfonate 1.80 3.00 3.00

NaFormate 0.08 0.08 0.08

NaOH 6.70 3.80 3.80

Silicon anti-foam agent 1.16 1.18 1.18

Ala216Glu 0.0145 - -

Ala216Glu + Tyr217Leu - 0.0145 -

Gln206Glu + Ala216Glu + Tyr217Leu - - 0.0145

Lipase .200 .200 .200

Cellulase - 7.50 7.50

Soil release polymer 0.29 0.15 0.15

Anti-foam ing agents 0.06 0.085 0.085

Brightener 36 0.095 - -

Brightener 3 - 0.05 0.05

C12 alkyl benzenesulfonic acid 9.86 - -

C12-15 alkyl polyethoxylate (2.5) sulfate 13.80 18.00 18.00

C 2 glucose amide - 5.00 5.00

C 12-13 alkyl polyethoxylate (9) 2.00 2.00 2.00

Water, perfume and minors balance to 100% c. Bar fabric cleaning compositions

Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01 % to about 1 % by weight of the composition.

The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 33-36 Bar Fabric Cleaning Compositions

Example No.

Component 33 34 35 36

Val203Glu 0.3 - 0.1 0.02

Tyr214Phe + Tyr217Asn - 0.3 0.4 0.03

C 2-C 6 alkyl sulfate, Na 20.0 20.0 20.0 20.00

C12-C14 N-methyl glucamide 5.0 5.0 5.0 5.00

Cι ι-Cι₃ alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00

Sodium carbonate 25.0 25.0 25.0 25.00

Sodium pyrophosphate 7.0 7.0 7.0 7.00

Sodium tripolyphosphate 7.0 7.0 7.0 7.00

Zeolite A (0.1 -.10μ) 5.0 5.0 5.0 5.00

Carboxymethylcellulose 0.2 0.2 0.2 0.20

Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20

Coconut monethanolamide 5.0 5.0 5.0 5.00

Brightener, perfume 0.2 0.2 0.2 0.20

CaSθ4 1.0 1.0 1.0 1.00

MgSθ4 1.0 1.0 1.0 1.00

Water 4.0 4.0 4.0 4.00

Filler* balance to 100%

*Can be selected from convenient materials such as CaC0₃, talc, clay, silicates, and the like.

In Example 33, the BPN' variants recited in Table 2, among others, are substituted for Val203Glu, with substantially similar results.

In Example 34, the BPN' variants recited in Table 2, among others, are substituted for Tyr214Phe + Tyr217Asn, with substantially similar results. In Examples 35-36, any combination of the BPN' variants recited in Table 2, among others, are substituted for Val203Glu and Tyr214Phe + Tyr217Asn, with substantially similar results.

While particular embodiments of the subject invention have been disclosed, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BRODE, PHILIP F.

BARNETT, BOBBY L. RUBINGH, DONN N. GHOSH, CHANCHAL K.

(ii) TITLE OF INVENTION: SERINE PROTEASE CONTAINING FABRIC CLEANING COMPOSITIONS

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY

(B) STREET: 11810 EAST MIAMI RIVER ROAD

(C) CITY: ROSS (D) STATE: OH

(E) COUNTRY: USA

(F) ZIP: 45061

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: ROOF, CARL J.

(B) REGISTRATION NUMBER: 37,708

(C) REFERENCE/DOCKET NO. 5232 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 513-627-0081

(B) TELEFAX: 513-627-0260

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 275 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

Ala Gin Ser Val Pro Tyr Gly Val Ser Gin He Lys Ala Pro Ala Leu 1 5 10 15

His Ser Gin Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val He Asp 20 25 30

Ser Gly He Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gin Asp Asn Asn Ser His 50 55 60

Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser He Gly 65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gin Tyr Ser Trp He He Asn Gly He Glu

100 105 110

Trp Ala He Ala Asn Asn Met Asp Val He Asn Met Ser Leu Gly Gly 115 120 125

Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140

Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160

Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val He Ala

165 170 175

Val Gly Ala Val Asp Ser Ser Asn Gin Arg Ala Ser Phe Ser Ser Val

180 185 190

Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser He Gin Ser Thr 195 200 205

Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu He Leu Ser Lys His Pro Asn 225 230 235 240

Trp Thr Asn Thr Gin Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu He Asn Val Gin Ala

260 265 270

Ala Ala Gin 275

Claims

Claims:

1. A fabric cleaning composition comprising:

(a) an effective amount of a BPN¹ variant comprising wild-type amino acid sequence wherein the wild-type amino acid sequence at one or more of positions 1 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219 or 220 is substituted, characterized in that i. when a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala,

His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; ii. when a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr,

Pro, Gly, Gin, Asn, Ser, Asp or Glu; iii. when a substitution occurs at position 201, the substituting amino acid for position 201 is Gly, Gin,

Asn, Ser, Asp or Glu; iv. when a substitution occurs at position 202, the substituting amino acid for position 202 is Pro, Gin,

Asn, Ser, Asp or Glu; v. when a substitution occurs at position 203, the substituting amino acid for position 203 is Met, Cys,

His, Pro, Gly, Gin, Asn, Ser, Asp or Glu; vi. when a substitution occurs at position 204, the substituting amino acid for position 204 is Glu; vii. when a substitution occurs at position 205, the substituting amino acid for position 205 is Leu, Met,

Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or

Glu; viii. when a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn or

Ser; ix. when a substitution occurs at position 207, the substituting amino acid for position 207 is Asp or Glu; x. when a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly,

Gin, Asn, Ser, Asp or Glu; xi when a substitution occurs at position 209, the substituting amino acid for position 209 is lie, Val,

Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; xii when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gin,

Asn, Ser, Asp or Glu; xiii. when a substitution occurs at position 211, the substituting amino acid for position 211 is Ala, Pro,

Gin, Asn, Ser, Asp or Glu; xiv when a substitution occurs at position 212, the substituting amino acid for position 212 is Gin, Ser,

Asp or Glu; xv. when a substitution occurs at position 213, the substituting amino acid for position 213 is Trp, Phe,

Tyr, Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin,

Asn, Ser, Asp or Glu; xvi. when a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu,

He, Nal, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu; xvii. when a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro,

Gin, Asn, Ser, Asp or Glu; xviii. when a substitution occurs at position 216, the substituting amino acid for position 216 is His, Thr,

Pro, Gly, Gin, Asn, Ser, Asp or Glu; xix. when a substitution occurs at position 218, the substituting amino acid for position 218 is Glu; xx. when a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gin,

Asn, Ser, Asp; or Glu; and xxi. when a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly,

Gin, Asn, Asp or Glu; characterized in that the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'; and (b) one or more cleaning composition materials compatible with the BPN' variant.

2. The fabric cleaning composition of Claim 1, characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Asn or Ser; b. when a substitution occurs at position 211, the substituting amino acid for position 21 1 is Pro, Gin, Asn, Ser, Asp or Glu; c. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu; and d. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gin, Asn, Ser, Asp or Glu.

3. The fabric cleaning composition of Claim 2, characterized in that when position 216 is substituted, Gly is substituted for Ala at position 216.

4. The fabric cleaning composition of Claim 2, characterized in that when a substitution occurs at one or more of positions 199, 200, 201, 202, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 or 220, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 219 or 220 is Asp or Glu; and when a substitution occurs at one or both of positions 204 or 208, the substituting amino acid for positions 204 or 218 is Glu; and wherein a substitution preferably occurs at one or more of positions of 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212 or 215, more preferably at one or more of positions 200, 201, 202, 205 or 207.

5. The fabric cleaning composition of Claim 1 having a single amino acid substitution characterized in that the substitution is: a. Glu for Ala at position 216; b. Asp for Ala at position 216; or c. Glu for Val at position 203.

6. A fabric cleaning composition comprising: (a) a BPN' variant comprising wild-type amino acid sequence characterized in that the wild-type amino acid sequence at two or more of positions 199, 200. 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 219 or 220 is substituted, characterized in that i. when a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala,

His, Thr, Pro, Gly, Gin. Asn, Ser, Asp or Glu; ii. when a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr,

Pro, Gly, Gin, Asn, Ser, Asp or Glu; iii when a substitution occurs at position 201, the substituting amino acid for position 201 is Gly, Gin,

Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; vi when a substitution occurs at position 204, the substituting amino acid for position 204 is Asp or Glu; vii when a substitution occurs at position 205, the substituting amino acid for position 205 is Leu, Val,

Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; viii when a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn,

Ser, Asp, or Glu; ix when a substitution occurs at position 207, the substituting amino acid for position 207 is Asp or Glu; x. when a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly,

Gin, Asn or Ser; xi when a substitution occurs at position 209, the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; xii. when a substitution occurs at position 210, the substituting amino acid for position 210 is Ala, Gly,

Gin, Asn, Ser, Asp or Glu; xiii. when a substitution occurs at position 211, the substituting amino acid for position 211 is Ala, Pro,

Gin, Asn, Ser, Asp or Glu; xiv. when a substitution occurs at position 212, the substituting amino acid for position 212 is Gin, Ser,

Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly,

Gin, Asn, Ser, Asp or Glu; xvi. when a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu,

He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn or

Ser; xvii. when a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro,

Pro, Gly, Gin, Asn, Ser, Asp or Glu; xix. when a substitution occurs at position 217, the substituting amino acid for position 217 is Leu, He,

Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser,

Asp or Glu; xx. when a substitution occurs at position 218, the substituting amino acid for position 218 is Gin, Ser,

Asp or Glu; xxi. when a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu; and xxii. when a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Ser, Asp or Glu; characterized in that the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'; and (b) one or more cleaning composition materials compatible with the BPN' variant.

7. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at two positions.

8. The fabric cleaning composition of Claim 7 characterized in that the two substitutions are: a. Ala for Pro at position 210 and Thr for Gly at position 215; b. Phe for Tyr at position 214 and Asn for Tyr at position 217; c. Glu for Ala at position 216 and Leu for Tyr at position 217; d. Leu for He at position 205 and Glu for Ala at position 216; e. Leu for He at position 205 and Asp for Ala at position 216; f. Glu for Gin at position 206 and Glu for Ala at position 216; g. Asp for Ala at position 216 and Leu for Try at position 217; or h. Glu for Gin at position 206 and Leu for Try at position 217.

9. The fabric cleaning composition of Claim 7, characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Glu, Asn or Ser; b. when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gin, Asn, Ser, Asp or Glu; c. when a substitution occurs at position 211, the substituting amino acid for position 211 is Pro, Gin, Asn, Ser, Asp or Glu; d. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu; and e. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gin, Asn, Ser, Asp or Glu.

10. The fabric cleaning composition of Claim 6, characterized in that when a substitution occurs at positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219 or 220, the substituting amino acid for any of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219 or 220 is Asp or Glu; and when a substitution occurs at position 213, the substituting amino acid for position 213 is Asp; and wherein a substitution preferably occurs at two or more of positions 199, 200, 201, 202, 205, 207, 208, 209, 210, 211, 212, or 215, more preferably a substitution occurs at two or more of positions 200, 201, 202, 205 or 207.

11. The fabric cleaning composition of Claim 6, characterized in that Glu or Asp is substituted for Ala at position 216 and Leu is substituted for Tyr at position 217.

12. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at three positions.

13. The fabric cleaning composition of Claim 12 wherein the three substitutions are: a. Pro substituted for Gin at position 206, Ala substituted for Gly at position 211, and Glu substituted for Ala at position 216; b. Val substituted for He at position 205, Ala substituted for Pro at position 210, and Glu substituted for Lys at position 213; or c. Glu substituted for Gin at position 206, Glu substituted for Ala at position 216, and Leu for Tyr at position 217.

14. The fabric cleaning composition of Claim 12 characterized in that a. when a substitution occurs at position 206, the substituting amino acid for position 206 is Asn or Ser; b. when a substitution occurs at position 210, the substituting amino acid for position 210 is Gly, Gin, Asn, Ser, Asp or Glu; c. when a substitution occurs at position 211, the substituting amino acid for position 211 is Pro, Gin, Asn, Ser, Asp or Glu; d. when a substitution occurs at position 214, the substituting amino acid for position 214 is Leu, He, Val, Met, Cys, Ala, His, Pro, Gly, Gin, Asn, Asp or Glu; and e. when a substitution occurs at position 215, the substituting amino acid for position 215 is Pro, Gin, Asn, Ser, Asp or Glu.

15. The fabric cleaning composition of Claim 6, characterized in that the wild-type BPN' is substituted at four positions or five positions.

16. The fabric cleaning composition of Claim 15, wherein the substitutions are: a. Glu substituted for Val at position 203, Glu substituted for Gin at position 206, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217; b. Glu substituted for Val at position 203, Ala substituted for Pro at position 210, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217; c. Glu substituted for Val at position 203, Glu substituted for Gin at position 206, Thr substituted for Gly at position 215, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217; or d. Glu substituted for Val at position 203, Ala substituted for Pro at position 210, Thr substituted for Gly at position 215, Glu substituted for Ala at position 216, and Leu substituted for Tyr at position 217.

17. The fabric cleaning composition of any of Claims 1 through 16, characterized in that said composition is in the form of a liquid.

18. The fabric cleaning composition of any of Claims 1 through 16, wherein the composition comprises at least about 5% surfactant and at least about 5% builder, by weight of the composition.

19. A method for cleaning fabric, said method comprising contacting a fabric in need of cleaning with the composition of any of Claims 1 through 18.