CN1301813A - Engineering fungus containing glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene - Google Patents

Abstract

The present invention determines GL-7ACA acylatase gene complete sequence from Pseudomonas sq.130 strain and the zymoprotein in codes, provides the recombinant plasmid pMR24 containing the said gene and constitute E.cOli MMR204 as gene engineering host bacteria, which is different from original host bacterial with Beta-lactamase having destroying defecton CPC and its derivative. The host bacteria may be used in constituting gene engineering bacteria for treating CPC and its derivative. The present invention establishes a plant of GL-7ACA acylatase gene engineering bacteria with industrial application prospect of producing 7ACA by using GL-7ACA as intermediate.

Description

The engineering bacteria that contains glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene

The present invention relates to the cynnematin acylase; more particularly relate to Glularyl-7-amino-cephalo-alkanoic acid acylase (the Glutaryl-7-Amino-Cephalosporin Acid Acylase of pseudomonas Pseudomonassp.130 bacterial strain; GL-7ACA Acylase; the GL-7ACA acylase) complete sequence of gene and coded zymoprotein thereof, this gene clone in intestinal bacteria stably express and make the structure that cynnematin transforms the engineering host bacterium E.coliMMR204 make up for adaptation.

Mainly (Cephalosporin C, produced, but its processing condition are comparatively harsh, and have environmental pollution problems by cephalosporin by chemical cracking CPC) in the past for 7-ACA (7-amino-cephalosporanic acid).This just impels people constantly to explore the approach of producing 7-ACA with bioconversion method.Long-term come, the research of cynnematin acylase is subjected to the attention of biologist and pharmacy man, result of study demonstrates its important industrial value gradually.The scission reaction of the acyl side-chain in main catalysis CPC of cynnematin acylase and other the derivative molecular, character according to enzyme reaction substrate, this fermentoid is divided into two types again: (1) makes the direct acidylate of 7-bit amino hexanedioyl side chain of CPC, one step generated 7-ACA, and this kind of enzyme is called the CPC acylase; (2) (Glutaryl-7-Amino-Cephalosporin Acid GL-7ACA) obtains 7-ACA for main substrate is hydrolyzed to react, and this fermentoid is called the GL-7ACA acylase with glutaryl-7ACA.Utilize the cynnematin acylase that CPC is carried out the existing report of single stage method that direct Enzymatic transformation generates 7-ACA, still, the conversion vigor of CPC acylase is extremely low, still is difficult to enter the industrial application stage.Since the eighties, some results of study show that CPC can be by D-amino-acid oxidase (DAO) oxidative deamination of multiple biogenetic derivation, and the step of going forward side by side carries out decarboxylic reaction and generates GL-7ACA.The latter's 7-position side chain can be obtained 7-ACA by the hydrolysis of GL-7ACA acylase (ACY) institute.Two step enzyme method technologies by CPC generation 7-ACA under DAO and the GL-7ACA ACY katalysis are gradually ripe.The separation of relevant GL-7ACA acylase bacterium producing multi enzyme preparation, evaluation, zymologic property and the application in bio-transformation thereof and in recent years the further investigation of enzyme gene is also had many reports.Matsuda etc. measure J.Bacteriology 169:4821 such as (, 1987) Matsuda to having made all-cis preface for cynnematin acylase I and II encoding gene in Pseudomonas SE 83 bacterial strains.Aramori etc. have analyzed the gene order (J.Fermentation and Bioengineering 72:232-243,1991) of the GL-7ACA acylase of Pseudomonas A-14 bacterial strain.Also have in addition PseudomonasGK 16 J.Bacteriology 163:1222-1228 such as (, 1985) Matsuda and C427 (Ishii Y et al.J.Ferment.Bioeng., 1994,77:591) in the partial dna sequence report of GL-7ACA acylase gene.Chinese patent " step, two enzyme process were made 7-amino-cephalosporanic acid (CN 1104255A; the nineteen ninety-five) " Enzymatic transformation to CPC is described, but finds that in the process that makes up GL-7ACA acylase gene engineering bacteria the recombinant plasmid pMR10 that includes the acy gene segment can not stablize preservation in intestinal bacteria A-56 (E.coli A-56) host bacterium.Simultaneously, have the ampC gene on the karyomit(e) of e. coli host cell, the β-Nei Xiananmei of this coded by said gene (cephalosporinase) has extremely strong hydrolysis vigor to the amido linkage of beta-lactam nucleus in the CPC molecule; Though when carrying out the technology of a step two enzyme process manufacturing 7ACA with whole cell, can overcome the destruction activity of β-Nei Xiananmei to improve the generation productive rate of 7-ACA by adding beta-lactamase inhibitor Su Batanna (Salbactam), but because production cost and quality product, adding inhibitor is not the ideal measure.

The objective of the invention is: 1. the host bacterium of pair GL-7ACA acylase gene recombinant plasmid is transformed, and new genetically engineered host bacterium is provided, to overcome the destruction of cephalosporinase (β-Nei Xiananmei) to CPC and derivative thereof.This host bacterium can be general to be applied to handle CPC and derivative (as GL-7ACA and 7ACA) thereof be the structure of the genetic engineering bacterium of purpose.2. measure and derive from the GL-7ACA acylase gene complete sequence of Pseudomonas sp.130 bacterial strain and coded zymoprotein thereof.3. the entrained GL-7ACA acylase gene of above-mentioned Pseudomonas sp.130 bacterial strain recombinant plasmid pMR10 is transformed, provide new recombinant plasmid, to overcome instable shortcoming.1.pMR 24 construction of recombinant plasmid:

The invention provides a kind of new recombinant plasmid pMR24, it is a double-stranded circular DNA, and length is 8010bp, and its oligogene type is acy, amp, par.

Original GL-7ACA acylase gene recombinant plasmid pMR10 (Liu Yang Yun etc. that derive from Pseudomonas 130 bacterial strains; biotechnology journal 7:94-107 1991 8:15-22 1992) in E.coliA56 host, can not stablize preservation; go down to posterity under the no selective pressure condition, plasmid promptly can be lost from the host.When E.coli A56 (pMR10) can not detect the existence of plasmid through switching (suitable 10 generations) most filial generation bacterium colony that goes down to posterity once in the substratum that does not add penbritin.When a factor of decision recombinant plasmid stability was parental cell generation division, allocation base had certain effect because of (par) in the distribution process of plasmid DNA molecule cell.Be to improve the stability of pMR10 plasmid in E.coli A56 host, press Fig. 1 program par gene recombination acy gene downstream in the pMR10 plasmid.At first, the pMR10 plasmid is with restriction restriction endonuclease HpaI enzymolysis, and handles Molecular Cloning 1986 such as () Sambrook, plasmid molecule self connection of phosphate group to prevent enzymolysis of removing 5 ' end with calf intestinal phosphatase esterase (CIP).Cut by Hpa I enzyme that pEC302 is the donor plasmid of par group in the fragment blunt end of generation, this plasmid DNA is behind BamH I and EcoR I double digestion, par gene fragment through gel electrophoresis and electroelution recovery 0.45kb, be connected for making this fragment carry out tack with the pMR10 of above-mentioned Hpa I enzymolysis, with archaeal dna polymerase the sticky end of 0.45Kb par gene fragment is mended flat, the pMR 10 of the HpaI enzymolysis that is obtained with mend an amount of mixing of flat 0.45Kb par gene fragment and be connected with the T4DNA ligase enzyme.This connects mixture in order to Transformed E .coli A56 competent cell, be coated on the LB flat board that adds the 50ug/ml penbritin, to the single bacterium colony that occurs separate, the quick extracting of purifying and plasmid DNA and enzyme are cut evaluations Molecular cloning such as (, 1986) Sambcook acquisition recombinant plasmid pMR24.

The comparison of pMR24 and the pMR10 stability in E.coli A56: recombinant plasmid pMR 10 and pMR 24 all have resistance selective marker amp (amicillin resistance), therefore, energy normal growth in the cultured solution of broth that contains 50 μ g/ml penbritins, when used substratum does not contain microbiotic, because plasmid instability, in the process that goes down to posterity, part cell can lose plasmid, this part cell has also just been lost at the energy for growth that contains on the microbiotic substratum, and its proportion has promptly been represented the extent of stability of plasmid.The bacterium colony that goes down to posterity in the no medicine nutrient solution is carried out single bacterium colony to be separated.Then with the dibbling simultaneously of single bacterium colony to pastille with not on two kinds of flat boards of pastille, if all grow bacterium colony on two kinds of flat boards, illustrate that this cell still has plasmid; If the bacterium of institute's dibbling only bacterium colony occurs on the flat board of no medicine, do not grow and on the flat board of pastille, have, represent that then this bacterium has been taken place by plasmid lose.In view of the above, the stability to recombinant plasmid pMR24 and pMR10 detects.Simultaneously, the ability that has also compared the product acylase of culture.The result is as shown in table 1.As seen from Table 1 with pMR10 plasmid rather unstable in E.coli A56 of acylase gene, in not containing antibiotic nutrient solution,, after the bacterium colony with resistance has reduced to 37%, 20 generation of sum, only save as 4% through 10 fissiparities from generation to generation.And the E.coli A56 The stability of strain that has pMR24 increases, and breeds the antibiosis colony number through 20 generations and still keeps 88%.This shows that the par gene has obviously improved the stability of carrying the acylase gene recombinant plasmid.And the stability of producing acylase improves relatively.(table 1)

The relative percentage ratio of table 1 stability with enzymic activity

Bacterial strain	Growth from generation to generation	Plasmid stability		GL-7ACA acylase vigor
								Tested colony number	The resistance colony number	U/L	Enzyme is lived and is kept %
E.coliA56 (pMR24)	The preceding contrast of going down to posterity	????100	????100	?253	????100
							????10	????100	????86	?236	????93.3
	????20	????100	????88	?226	????89.2
							????30	????100	????61	?176	????69.5
?E.coliA56 (pMR10)	The preceding contrast of going down to posterity	????100	????100	????184	????100
							????10	????100	????37	????73	????39.7
	????20	????100	????4	????5	????2.7
							????30	????100	????3	????2.5	????1.4

2. derive from the complete sequence of GL-7ACA acylase gene of Pseudomonas sp.130 bacterial strain and coded zymoprotein thereof:

The GL-7ACA acylase gene acy that derives from Pseudomonas sp.130 bacterial strain is a double-stranded DNA, and length is 2482bp (seeing SEQID NO:1).

(1) subclone and determined dna sequence

Adopt subclone and two kinds of methods of synthetic primer, checking order used is the sequencing kit of Amersham company.

From pMR24, be that the subclone schema of the acy gene fragment of vector construction is seen Fig. 2 with pBluescript SK II (Stratagene company), the mensuration of sequence sees Table 2, and SEQ ID NO:3-10 has provided the sequence of the primer.

Used primer when table 2 subclone and the sequence surveyed thereof and order-checking

Plasmid	Make up	Order-checking	Primer
				???Pblue3	?PBluescript?SKⅡ/BamHI+B2→B1?(α)	BamHⅠ2→BamHⅠ1	M13,KS
???Pblue4	?PBluescript?SKⅡ/BamHI+B1→B2(α)	BamHⅠ1→BamHⅠ2	M13,KS,α
				???Pblue7	?PBluescript?SKⅡ/BamHI+B3→B2(β)	BamHⅠ2→XhoⅠ	M13,KS,β1
???Pblue8	?PBluescript?SKⅡ/BamHⅠ+B2→B3(β)
				???Pblue15	?PBlue8/XhoⅠ?B2→XhoⅠ(f)	XhoⅠ→BamHⅠ2	?Reverse,SK
???Pblue16	?PBlue7/XhoⅠ?B3→XhoⅠ(f)	XhoⅠ→MluⅠ	Reverse,SK,β4
				???Pbluel8	?PBlue8/Mlu,EcoRV,Klenow?B2→MluⅠ(f)	MluⅠ→XhoⅠ	Reverse,SK,β3
???Pblue19	?PBlue7/Mlu,EcoRV,Klenow?B3→MluⅠ(f)	MluⅠ→stop?codon	?Reverse,SK

(2) mensuration of GL-7ACA acylase N end and c terminal amino acid sequence reaches with the aminoacid sequence of similar acylase and compares

A kind of GL-7ACA acylase of pseudomonas Pseudomonas sp.130 bacterial strain that derives from is by α; two subunits of β are formed; the signal peptide of zymoprotein is made up of 33 amino acid (MetLeuArgValLeuHisArgAlaAlaSerAlaLeuValMetAlaThrValIleGl yLeuAlaProAlaValAlaPheAlaLeuAlaGluProThrSer) or 40 amino acid (MetLeuArgValLeuHisArgAlaAlaSerAalaLeuValMetAlaThrValIleG lyLeuAlaProAlaValAlaPheAlaLeuAlaGluProThrSerThrProGlnAla ProIleAla); connection peptides is made up of 10 amino acid (GlyAspProProAspLeuAlaAspGlnGly); for the result to the dna sequencing of the zymoprotein of Pseudomonas sp.130 GL-7ACA acylase gene acy provided by the invention coding verifies; to the N end of the α subunit of zymoprotein, the N terminal amino acid sequence of C end and β subunit checks order.Sequencing result shows that the nucleotide sequence and the aminoacid sequence that are provided in Pseudomonassp.130 GL-7ACA acylase nucleotide sequence provided by the invention and aminoacid sequence (seeing SEQ ID NO:1 or SEQ IDNO:2) and the U.S. Pat 05457032 are identical.The GL-7-ACA acylase aminoacid sequence height homology (sequence is relatively seen Fig. 3 and Fig. 4) that derives from Pseudomonas sp.GK-16 and C427 with bibliographical information; from aminoacid sequence GK-16 more as can be seen, the acylase among C427 and the present invention has constituted the acylase family with GL-7ACA hydrolysis vigor.

The result of enzymology shows; the enzyme of acylase of the present invention (13U/mg albumen) alive is higher than the enzyme (＜10U/mg albumen) alive of GK-16 and other similar acylases; the KM value (0.5mM) of acylase of the present invention is higher than GK-16 acylase (0.16mM), but be lower than other similar acylases (＞1mM).Therefore, but the structure of enzyme of the present invention is similar distinguishing to the acylase of having delivered from GK-16 and C427 to catalysis characteristics.3. make up E.coli MMR 204 genetically engineered host bacterium

Adopt external fixed zone insertion sudden change and the interior recombinant technology of body to make up genetically engineered host bacterium E.coliMMR204, its oligogene type is ampC ∷ KAPA, ompT, recA, used bacterium, phage and plasmid see Table 3, make up process program (seeing Fig. 5, Fig. 6) and are described in detail as follows:

(1) pZJ2 of plasmid, the acquisition of pZJ3 (Fig. 6)

With the ampC gene on the plasmid pJAC-4 is template, two pairs of oligonucleotide Nde I 5 ' and EcoR I 3 ', EcoR I 5 ' and BamH I 3 ' be primer, utilize round pcr, amplification obtains ampC5 ' Nde I/EcoR I fragment and ampC3 ' EcoR I/BamH I fragment, cut with EcoR I enzyme respectively again and connect,, can obtain having the ampC fragment in EcoR I site through the gel electrophoresis separation and purification.AmpC EcoR I fragment is connected with carrier pGEM-T, is transformed into DH10B, on LB Amp flat board, select the bacterium colony of lacZ, can obtain fragment direction of insertion opposite recombinant plasmid pZJ2 and pZJ3.

Table 3 bacterial strain, phage and plasmid

Bacterial strain, phage and plasmid	The related gene type	The source
			?DH10B	F-?mcrAΔ(mrr-hsdRMS-mcrBL)Φ80d?lacZ?ΔM15ΔlacX74 endA1?recA1?deoRΔ(ara,leu)7697?araD?139?galm?galk?nupG?rpsL	BRL company
?JC7623	recB,recC,sbcB	??Winans
			?AKK241	ampCp1,zie2241∷Tn10	??I.R.Booth
?BL21	F -?ompT?hsd?s R(r R -m B -)	??J.Sambrook
			?SP946	recA ,srl∷Tn10	??G.P.Zhao
?MMR200	JC7623?ampC∷KAPA	This work
			?MMR202	AKK241?ampC∷KAPA???????????????????????????????????????????	This work
?MMR203	BL21?ampC∷KAPA?????????????????????????????????????????????	This work
			?MMR204	MMR203?ampC∷KAPA	This work
?Plclg		??R.L.Somerville
			?PJAC-4	?ampC,kan	??Bengtake?Jaurin
?PUC4-KAPA	?KAPA	U.S. Pharmacia company
			?PGEM-T	?LacZ	U.S. Promega company
?pZJ2	AmpC EcoR I and lacZ in the same way, the pGEM-T carrier	This work
			?pZJ3	AmpC EcoR I and lacZ are reverse, the pGEM-T carrier	This work
?pZJ4	AmpC ∷ KAPA, KAPA and ampC in the same way, the pZJ2 carrier	This work
			?pZJ5	AmpC ∷ KAPA, KAPA and ampC are reverse, the pZJ2 carrier	This work

(2) plasmid pZJ4, the acquisition of pZJ5 (Fig. 6)

Utilize EcoR I enzyme to cut gel electrophoresis separation and electroelution, acquisition contains the EcoR I enzyme of that mycin resistant gene and cuts dna fragmentation KAPA, kalamycin resistance gene KAPA is inserted the pZJ2 that cuts through EcoR I enzyme, be transformed into DH10B, coat on the LB flat board that contains kantlex, in 37 ℃ of cultivations 24 hours, the resistance bacterium colony plasmid DNA that obtains is carried out enzyme with the EcoR I cut inspection.Obtain fragment direction of insertion opposite recombinant plasmid pZJ4 and pZJ5.

(3) acquisition of JC7623 ampC ∷ KAPA

Get pZJ5 DNA, add restriction enzyme Sca I, enzyme was cut two and a half hours, get linear DNA then and transform JC7623, select the kalamycin resistance transformant, and the responsive transformant of screening penbritin, the transformant of anti-kantlex is required JC7623ampC ∷ KAPA to penbritin is responsive, names to be MMR200.

(4) genetics and the biochemical evaluation (acquisition of bacterial strain MMR201 and MMR202 and evaluation thereof) of ampC ∷ KAPA sudden change in the MMR200 bacterial strain

P1 transduction method (the Jeffrey H.Miller of employing standard, A shout Course in BacteriaGenefics, A laboratory manual and handbook for Escherichia coli and relatedbacteria) the cynnematin enzyme activity of the transduttant of intestinal bacteria mutant strain AKK241 (24 μ/mg dry weight, table 4) 100% kalamycin resistance of the P1 lysate of MMR20 transduction high expression level ampC gene is but dropped to almost can't measure low-level (＜7 * 10 ^-4μ/mg dry weight, table 4).Because the chromogene ampCp1 of AKK241 and zje2241 ∷ Tn10 are chain, therefore the transduttant with kalamycin resistance further further screens on LB tsiklomitsin flat board, the tetracyclin resistance bacterial strain that obtains is named and is MMR201, the bacterial strain of sensitive tetracycline is named and is MM202, and wherein the ratio of sensitive strain is about 1%.This presentation of results KAPA and zje2241 ∷ Tn10 are chain.And the MM202 of sensitive tetracycline more can be used for further transformation.

(5) BL21 ampC ∷ KAPA obtains

Utilize proteolytic enzyme OmpT sudden change to make the foreign protein that is expressed in periplasmic space comparatively stable, the P1 lysate transduction BL21 (ompT defective) with MMR202 selects the kalamycin resistance transduttant, names to be MMR203.

(6) MMR203 is transform as the recA bacterial strain

Because the recA defective can guarantee plasmid stable going down to posterity and the stably express of clone gene, P1 lysate transduction MMR203 with SP946, select the tetracyclin resistance transduttant, utilize ultraviolet excess susceptibility screening and the closely linked recA deletion mutantion of srl ∷ Tn10 transduttant then, name and be MMR204.

(7) mensuration of ampC ∷ KAPA beta-lactam enzyme activity

Compare (table 4) with being integrated with the bacterial strain of gene ampC ∷ KAPA and the beta-lactam enzyme activity of former bacterial strain on the karyomit(e).AKK241 is an ampCp1 rising mutant strain, and high beta-lactam enzyme activity is arranged.Transform its beta-lactam enzyme activity of back than still measuring under the exacting terms.A56-3 has certain beta-lactam enzyme activity for our original host bacterium of adopting.The beta-lactam enzyme activity of BL21 is lower, lives but still can measure enzyme under the condition of concentrated 10 times of bacterium liquid, and improved beta-lactam enzyme activity also can't record.The data of table 5 are the mean value of three parallel laboratory tests.

The beta-lactam enzyme activity of some E.coli bacterial strains of table 4

Bacterial strain	The related gene type	Beta-lactam enzyme activity (μ/mg dry weight)
			???AKK241	????ampCp1,zje2241∷Tn10	????23.89+/-0.87
???A56-3	????F’trp?recA?ampC +	????1.03+/-0.09
			???BL21	????ampC +	????0.090+/-0.001
???MMR202	????AKK241?ampC∷KAPA	????＜7×10 -4
			???MMR203	????BL21?ampC∷KAPA	????＜4×10 -4
???MMR204	????MMR203recA?srl∷Tn10	????＜4×10 -4

4. former engineering bacteria A _56-3/ pMR24 and new construction bacterium MMR204/pMR24 are applied to GL-7ACA comparison to the destructiveness of beta-lactam nucleus in the conversion process of 7ACA

New construction bacterium MMR204/PMR24 is by the common micro-organisms center preservation of Beijing China Committee for Culture Collection of Microorganisms, and on June 23rd, 1997, deposit number is CGMCC NO.0309.

With former engineering bacteria A _56-3/ pMR24 and new construction bacterium MMR204/pMR24 are applied to the conversion of GL-7ACA to 7ACA respectively, and relatively they are to the destructiveness (see Table 5, substrate GL-7ACA concentration is 0.133223mg/ml) of beta-lactam nucleus.

The former engineering bacteria A of table 5 _56-3/ pMR24 and new construction bacterium MMR204/pMR24 are applied to

GL-7ACA in the conversion process of 7ACA to the destructiveness of beta-lactam nucleus

Engineering bacteria	Reaction back 7ACA (mg/ml)	Reaction back GL-7ACA (mg/ml)	The destruction of beta-lactam nucleus (%)
				??A 56-3/pMR24	??????0.043778	???????0.028234	??????????32.3
??MMR204/pMR24	??????0.055199	???????0.038581	??????????12.2

From the data of table 5 as can be seen, improved engineering bacteria reduces greatly to the destructiveness of beta-lactam nucleus, can be applicable among large-scale the production.

Use the mutant clon that extracorporeal recombination makes up a certain gene, by reorganization in the body sudden change is integrated into karyomit(e) then and is called as " reverse genetics ", be the method that the present invention takes with the method for the new organism that makes up this transgenation.Along with the development of molecular biosciences and bio-engineering research work, this method will show its great potential day by day and be noted widely.The present invention has successfully used above-mentioned technique construction E.coli MMR204 bacterial strain, and function aspects carried out bacterial strain transform before and after the comparison of beta-lactam enzyme activities, from the data of table 4 as can be seen, all E.coli P1 transduttants that has ampC ∷ KAPA sudden change, no matter the height of its parental plant beta-lactam enzyme activity all loses the ability of synthesizing this enzyme fully.Simultaneously, in MMR204 directly applies to conversion process from GL-7ACA to 7ACA as the host bacterium, find that the beta-lactam structure of GL-7ACA and 7ACA remains stable.Therefore, it is the structure of goal gene engineering bacteria to handle CPC and derivative (as GL-7ACA or 7ACA) thereof that this host bacterium can usually be applied to all, promptly as a kind of cephalosporinase (being the ampC defective) that do not produce, can keep that plasmid is stable to go down to posterity, guarantee clone gene stably express (recA defective), periplasmic space proteolytic enzyme defective (ompT defective), the host bacterium of being convenient to transform that extensive use is arranged.

Advantage of the present invention: glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene of the present invention and engineering bacteria thereof, a kind of new recombinant plasmid pMR24 is provided, overcome the unstable of original recombinant plasmid pMR10; Measured a kind of Pseudomonas sp.GK-16 and the GL-7-ACA acylase gene complete sequence C427 acylase, that derive from Pseudomonas sp.130 bacterial strain and coded zymoprotein of having delivered thereof that be different from; And provide a kind of new genetically engineered host bacterium MMR204, the β-Nei Xiananmei that has overcome original host bacterium is to CPC and the damaging shortcoming of derivative thereof, and this host bacterium can be general, and to be applied to handle CPC and derivative (as GL-7ACA and 7ACA) thereof be the structure of the genetic engineering bacterium of purpose.What the present invention had set up that a strain has a prospects for commercial application is the GL-7ACA acylase gene engineering bacteria that intermediate is produced 7ACA with GL-7ACA.

The present invention is further elaborated by the following drawings and embodiment, but does not limit the scope of the invention.

Description of drawings

Fig. 1. the structure of recombinant plasmid pMR24.

The subclone schema of Fig. 2 .acy gene fragment

B1=BamHⅠ1,B2=BamHⅠ2,B3=BamHⅠ3,Ev=EcoRⅤ,M=MluⅠ,P=PstⅠ,S=SalⅠ,X=XhoⅠ。

The comparison of Fig. 3 .Pseudomonas sp.130 GL-7-ACA and Pseudomonas sp.GK-16 acylase aminoacid sequence.

The comparison of Fig. 4 .Pseudomonas sp.130 GL-7-ACA and Pseudomonas sp.C427 acylase aminoacid sequence.

The structure process program of Fig. 5 .E.coli MMR204 bacterial strain

PZJ2:ampC EcoR I and lacZ in the same way, the pGEM-T carrier

PZJ3:ampC EcoR I and lacZ are reverse, the pGEM-T carrier

PZJ4:ampC ∷ KAPA, KAPA and ampC in the same way, the pZJ2 carrier

PZJ5:ampC ∷ KAPA, KAPA and ampC are reverse, the pZJ2 carrier

MMR202：AKK241?ampC∷KAPA

MMR203：BL241?ampC∷KAPA

MMR204：MMR203?recA?srl∷Tn10

Primer Nde I 5 ': design 5 ' end contains the 5 ' ACCAGACCATATGTTCAAA that draws in Nde I single endonuclease digestion site

Primer EcoR I 3 ': design 3 ' end contains the primer 5 ' CCCAGGCGGAATTCTTTTC in EcoR I single endonuclease digestion site

Primer EcoR I 5 ': design 5 ' end contains the primer 5 ' AAAAGAATTCCGCCTGGGG in EcoR I single endonuclease digestion site

Primer BamH I 3 ': design 3 ' end contains the primer 5 ' CCCAGGCGGAATTCTTTTC in BamH I single endonuclease digestion site

AmpC 5 ' Nde I/EcoR I: with Nde I 5 ' and EcoR I 3 ' be primer, pJAC-4 is a template, by the gene fragment that obtains behind the pcr amplification

AmpC 3 ' EcoR I/BamH I: with EcoR I 5 ' and BamH I 3 ' be primer, pJAC-4 is a template, by the gene fragment that obtains behind the pcr amplification

AmpC EcoR I: the ampC gene fragment in an EcoR I single endonuclease digestion site is contained in the centre

Fig. 6. contain the structure schema of the recombinant plasmid pZJ5 of ampC ∷ KAPA sudden change.

The reorganization of embodiment 1 GL-7ACA acylase gene plasmid and par gene fragment

The molecular biology method that relates in the present embodiment is all with reference to " Molecular Cloning--A LaboratoryManual " ed.by J.Sambcook, E.F.Fritsch and T.Maniatis, 1989.CSHL Press.

Step 1:par gene fragment is separated:

Extract the pEC302 plasmid DNA, behind the super centrifugal purification of CsCl density gradient, with BamH I and the complete double digestion of EcoR I, on 0.6% low melting-point agarose gel, electrophoresis reclaims the par gene fragment of 0.5Kb.

Step 2: the preparation of flush end par gene fragment

Getting 0.5Kb par gene fragment and the 2.5ul dNTP (2mM) that 2g step 1 obtains is mixed in the 50ul otch damping fluid, add 2.5u e. coli dna polymerase I Klenow fragment, 20 ℃ are incubated 30 minutes, 1ul 0.5 M EDTA termination reaction, at phenol, the chloroform (volume ratio 1: 1) of 50ul, each suction filtration of 50ul chloroform once.Reclaim flush end par gene fragment with the long-pending dehydrated alcohol precipitation of triploid again.

Step 3: through the dephosphorization of the pMR10 DNA of Hpa I digestion

On the pMR10 plasmid, the Hpa I point of contact in acylase gene downstream can be used as the insertion point of par gene fragment, because the Hpa I is that the flush end enzyme is cut; therefore; can be connected with the par gene fragment of flush end, connect, need slough its 5 ' end p with the joint efficiency of raising with the par gene fragment for reducing self.For this reason, get the pMR10 DNA of 5ug Hpa I digestion, be dissolved in the 50ul dedicated buffering liquid, add 0.05U Roll phosphoesterase, mixing, 37 ℃ were reacted 1 hour, with 300ul stop buffer (10mM Tris, 1mM EDTA, 200mM NaCl, 0.5%SDS) reaction is stopped, with step 2 method purifying and reclaim the Hpa I digestion pMR 10DNA of dephosphorization.

Step 4: the reorganization of linear pMR 10DNA and par gene fragment

The dephosphorization Hpa I pMR 10DNA 300ng (10ul) that is obtained by step 2 and step 3 mixes with flush end par gene fragment 150ng (3ul) respectively, precipitate through dehydrated alcohol, be dissolved in 10ul and connect in the damping fluid, add 1ul T4 dna ligase, 15 ℃ were reacted 16 hours.

Step 5: the screening of band par gene recombination plasmid pMR24

The ligase enzyme mixed solution of step 4 be used for Transformed E .coli A56 competent cell and the broth culture that contains the 50ug/ml penbritin (peptone: 1%, yeast powder: 0.5%, NaCl:1%, pH:7.0) dull and stereotyped going up obtains transformant.Further 12 transformed bacterias are dropped into the quick extracting of capable plasmid DNA and restriction enzyme digestion and electrophoresis is identified, determine to have in the plasmid DNA of four strain transformed bacterias the external source par gene fragment of 0.5Kb, this recombinant plasmid is named and is pMR24.

The stability analysis (the results are shown in Table 1) of embodiment 2 recombinant plasmid pMR24 in host bacterium E.coli A56:

The continuous passage of step 1:E.coli A56 (pMR24)

By embodiment 1, the E.coli A56 (pMR24) that step 5 obtains is inoculated into the 3ml broth culture, and (composition is seen embodiment 1, in the test tube of 10 * 150mm of step 5), places on 100 rev/mins of shaking tables 37 ℃ to cultivate 8-12 hour.Get this nutrient solution 30 ul and be transferred in the above-mentioned fresh broth culture test tube, cultivate and will once pass with method and be estimated as 10 generations of bacterial reproduction the vegetative period that connects.Bacterial strain E.coli A56 (pMR10) cultivates with method in contrast.

Step 2: recombinant plasmid Detection of Stability

After three switchings are gone down to posterity in the step 1, respectively to E.coli A56 (pMR10) and E.

The cultivation medicine of coli A56 (pMR24) carries out 10 ^-6, 10 ^-7, 10 ^-8Dilution, getting 0.1ml dilution bacterium liquid is applied on the broth culture flat board, with the single bacterium colony that obtains, further with aseptic toothpick with the dibbling simultaneously of single bacterium colony to contain Amp (50ug/ml) broth culture dull and stereotyped with no medicine broth culture flat board on, 37 ℃ of overnight incubation, the resistance colony number on the record Amp flat board.

Step 3:GL-7ACA acylase vitality test

E.coli A56 (pMR 10) and E.coli A56 (pMR 24) are in the process that goes down to posterity of step 1, get nutrient solution 0.5ml, 4000 rev/mins centrifugal 10 minutes, abandon supernatant liquor, thalline is with 0.5ml phosphoric acid buffer (0.1M, pH7.0) after the suspension, add 50mg/ml GL-7ACA 0.5mL, 37 ℃ of water bath heat preservations 30 minutes are used the 3ml[20% Glacial acetic acid, 0.05N NaOH (2: 1)] the stop buffer termination reaction, and 0.5% pair of methylamino phenenyl formaldehyde of adding 0.5ml makes reaction product 7ACA colour developing.Recentrifuge 15 minutes, supernatant liquor is in 415nm place photometry absorption value.The definition of 1u GL-7ACA acylase vigor: (37 ℃, 0.1M pH7.0 phosphoric acid buffer) per minute can transform GL-7ACA and generate the enzyme amount of 1ug7-ACA under given conditions.

That is: enzyme activity (U/ml)=K * OD 415nm/ (t * D)

The K:7-ACA slope of standard curve

T: enzyme reaction time

D: the volume that adds bacterium liquid in the reactive system

The mensuration (sequence is seen SEQ IDNO:2) of embodiment 3 GL-7ACA acylase N terminal amino acid sequences

(Hermann?S?and?Gebhard?VJ(1987).Anal?Biochem.166:368-379)

Step 1: electroblotting step

(1) the Hyperbond film is immersed in about 1min in the methyl alcohol, then film being moved to the 10mM CAPS that contains 20ug/mlDTT and 10% methyl alcohol changes in the film damping fluid, be saved in be used before.

(2) the GL-7-ACA acylase sample behind the purifying carries out TSDS-PAGE electrophoresis (the thick 0.5mm of gel) with reference to the method for (1987) such as Hermann, and the gel behind the electrophoresis is carefully dialled down, puts into to contain 20

Rinsing in the redistilled water of ug/ml DTT, then gel being soaked in the 10mM CAPS that contains 20ug/ml DTT and 10% methyl alcohol changes about 10min in the film damping fluid (changing a damping fluid every 5min).

(3) gel and Hyperbond film are fixed by the mode of " Layer cake ", and put into membrane-transferring device.Under 4 ℃, constant voltage 50V electrotransfer 1h.

(4) the Hyperbond film is taken out from membrane-transferring device, and put into the redistilled water rinsing that contains 20ug/ml DTT.

(5) the Hyperbond film that will shift sample is put into methanol/acetate=5/4/1 dissolved 2mg/ml and is examined the Ma Shi light blue R-250 solution 30min that dyes, and decolours in the solution of methanol/acetate=5/4/1 then, till background is white.

Step 2: amino acid sequence analysis

The protein protomer of preparing order-checking is cut down from the Hyperbond film, and put on the BeckmanLF-3200 protein polypeptide order instrument and measure the N terminal amino acid sequence.

The mensuration (sequence is seen SEQ IDNO:2) of embodiment 4 GL-7-ACA acylase α subunit C terminal sequences

(1) the GL-7-ACA acylase to purifying carries out the SDS-PAGE electrophoresis.

(2) take off gel from electrophoresis apparatus, place 0.25MKCl solution (4 ℃), can see the α and the β subunit that separate on the gel clearly, downcut the α subunit with scalpel, and cut into pieces.

(3) fragment is put into the electroelution device, constant current 8-10mA, wash-out are about 8 hours, and taking-up contains the elutriant of α subunit in the eppendorf pipe.

(4) acetone (20 ℃) that adds 4 times of effluent volumes is in above-mentioned eppendorf pipe, ice bath 15 minutes, and 12000 left the heart 10 minutes, abandoned supernatant, the precipitation dissolved in distilled water, and with distill water dialysis three times.

(5) dialyzate is measured the C terminal sequence with TEABI procise-491 type protein C end sequenator.

The structure (seeing Fig. 5, Fig. 6) of embodiment 5 GL-7ACA acylase engineering host bacterium MMR204

In the present embodiment, plasmid extraction, enzyme are cut, are connected, conversion and PCR method, all basis

J.Sambrook?et?al,Molecular?Cloning。

P1 phage transduction method, according to Jeffrey H.Miller, A shout Course in BacteriaGenetics, A laboratory manual and handbook for Escherichia coli and relatedbacteria. restriction endonuclease is a Promega company product.

Substratum and antibiotic consumption: LB substratum, the R substratum, the R semisolid medium, all with Jeffrey H.Miller, A shout Course in Bacteria Genetics, A laboratory manual andhandbook for Escherichia coli and related bacteria.

Penbritin 100-200ug/ml; Kantlex 50-100ug/ml; Tsiklomitsin 50ug/ml; Streptomycin sulphate 50ug/ml;

Step 1: the utilization extracorporeal recombination obtains containing the recombinant plasmid pZJ5 of ampC ∷ KAPA sudden change

1. the pZJ2 of plasmid, the acquisition of pZJ3 (Fig. 6)

(1) be template with the ampC gene on the plasmid pJAC-4, the two pairs of oligonucleotide Nde I 5 ' and EcoR I 3 ', EcoR I 5 ' and BamH I 3 ' be primer, utilize round pcr, amplification obtains ampC5 ' Nde I/EcoR I fragment and ampC3 ' EcoR I/BamH I fragment, cut with EcoR I enzyme respectively again and connect,, can obtain having the ampC fragment in EcoR I site through the gel electrophoresis separation and purification.This fragment may go out three kinds of situations: the connection product I of two ampC5 ' Nde I/EcoR I; The connection product II of two ampC 3 ' EcoR I/BamH I; AmpC5 ' Nde I/EcoR I and ampC3 ' EcoR I/BamH I be connected the product III, because of two fragment ampC5 ' are close with ampC3 ' length, gel electrophoresis can't separate three kinds of junction fragments.

(2) gained fragment in (1) is connected with carrier pGEM-T, is transformed into DH10B, on LB Amp flat board, select the bacterium colony of lacZ.The entrained recombinant plasmid of these bacterium colonies may have three types.An EcoR I site is arranged, no BamH I site on the plasmid that pGEM-T and fragment I are connected to form; An EcoR I site is arranged on the plasmid that pGEM-T and fragment II are connected to form, two BamH I sites are arranged; An EcoR I site is arranged on the plasmid that pGEM-T and fragment III are connected to form, a BamH I site is arranged.Therefore, bacterium colony DNA is carried out the EcoR I respectively, BamH I enzyme is cut, and just can distinguish three types, obtains the required pGEM-T and the product that is connected of fragment III.

(3) pGEM-T and fragment III recombinant plasmid are carried out further enzyme and cut inspection, to determine to connect the closure of product ampC EcoRI.Because plasmid pGEM-T is on lacZ α structure gene, the multienzyme that is positioned at the foreign DNA 5 ' end of insertion is cut on the joint order of site a Pst I site, near BamH I end a Pst I site is also arranged and ampC EcoRI is segmental, so can be according to the size of Pst I endonuclease bamhi, obtain connecting back ampC EcoR I relative lacZ α gene plasmid pZJ2 in the same way, reverse plasmid pZJ3.

2. plasmid pZJ4, the acquisition of pZJ5 (Fig. 6)

Utilize EcoR I enzyme to cut gel electrophoresis separation and electroelution, acquisition contains the EcoR I enzyme of that mycin resistant gene and cuts dna fragmentation KAPA, kalamycin resistance gene KAPA is inserted the pZJ2 that cuts through EcoR I enzyme, be transformed into DH10B, coat on the LB flat board that contains kantlex, in 37 ℃ of cultivations 24 hours, the resistance bacterium colony plasmid DNA that obtains is carried out enzyme with the EcoR I cut inspection.Novel plasmid after more resulting KAPA being inserted carries out further enzyme and cuts inspection, with the direction of determining that KAPA inserts.Because an Xho I point of contact is being arranged, near Nde I end an Xho I point of contact is also arranged on the ampC EcoR I, and carrier pGEM-T goes up no Xho I point of contact, so can judge the direction that KAPA inserts according to the size of Xho I endonuclease bamhi near place, KAPA top.

Step 2: the recombinant technology ampC ∷ KAPA that will suddenly change is integrated into ampC gene locus on the escherichia coli chromosome in the utilization body, makes up to contain the bacterial strain that ampC ∷ KAPA suddenlys change

1.JC7623 the acquisition of ampC ∷ KAPA

Get 20 μ g pZJ5 DNA, add 20u Sca I, enzyme was cut two and a half hours, get 10 μ g linear DNAs then and transform JC7623, select the kalamycin resistance transformant, and the responsive transformant of screening penbritin, the transformant of anti-kantlex is required JC7623ampC ∷ KAPA to penbritin is responsive, names to be MMR200.

2.MMR200 the genetics and the biochemical evaluation (bacterium of ampC ∷ KAPA sudden change in the bacterial strain

The acquisition of strain MMR201 and MMR 202 and evaluation thereof)

P1 transduction method (the Jeffrey H.Miller of employing standard, A shout Course in BacteriaGenefics, A laboratory manual and handbook for Escherichia coil and relatedbacteria) the cynnematin enzyme activity of the transduttant of intestinal bacteria mutant strain AKK241 (24 μ/mg dry weight, table 4) 100% kalamycin resistance of the P1 lysate of MMR200 transduction high expression level ampC gene is but dropped to almost can't measure low-level ((7 * 10 ^-4μ/mg dry weight, table 4).Because the chromogene ampCp1 of AKK241 and zje2241 ∷ Tn10 are chain.Therefore the transduttant with kalamycin resistance further further screens on LB tsiklomitsin flat board, and the tetracyclin resistance bacterial strain that obtains is named and is that MMR201, the bacterial strain of sensitive tetracycline name and is M202, and wherein the ratio of sensitive strain is about 1%.This presentation of results KAPA and zje2241 ∷ Tn10 are chain.And the MM202 of the every sense of tsiklomitsin more can be used for further transformation.

Step 3: the last bacterial strain MMR204 that obtains to meet the engineering bacteria needs again through the transformation in some steps

1.BL21ampC ∷ KAPA obtains

Utilize the P1 lysate transduction BL21 (ompT) of MMR202, select the kalamycin resistance transduttant, name and be MMR203.

2. MMR203 is transform as the recA bacterial strain

P1 lysate transduction MMR203 with SP946 selects the tetracyclin resistance transduttant, utilizes screening of ultraviolet excess susceptibility and the closely linked recA deletion mutantion of srl ∷ Tn10 transduttant then, names to be MMR204.

The mensuration (data see Table 4) of embodiment 6 beta-lactam enzyme activities

1. the preparation of tested bacterium liquid: picking one single bacterium colony from the LB flat board, be inoculated in and be added with in the corresponding antibiotic LB nutrient solution, 37 ℃ of jolting overnight incubation, suitably dilution (claiming the OD extension rate) back is in OD ₆₀₀Survey bacteria concentration.(the OD value is between the 0.4-0.8);

2. prepare the 1mg/ml CPC-Na aqueous solution;

3. (looking the enzyme height of living suitably concentrates or dilution with being suspended in behind the tested bacterium liquid precipitate in the phosphoric acid buffer of PH=7.6, claim cycles of concentration or extension rate) and the preheating 10 minutes in 35 ℃ of waters bath with thermostatic control of CPC-Na solution, respectively get the equal-volume number then and mix insulation, take out the 1ml mixed solution respectively at different time and add (20% acetic acid: 0.05N NaOH=2: 1) in the 4ml stop buffer, add stop buffer earlier with after the bacterium liquid insulation, after add substrate and be contrast.Getting supernatant liquor after reaction solution and contrast liquid are centrifugal, is blank with the damping fluid, 260nm photometry density.

4. the calculating of living than enzyme: the enzyme work/0.5ml bacterium liquid than enzyme (u/mg)=0.5ml bacterium alive liquid contains dry cell weight

=[Δ OD ₂₆₀* enzyme is lived, and (or ÷ concentrates doubly coefficient * 10 * 60min * extension rate

Number)]/(OD ₆₀₀* dried fungus strain number * OD extension rate * 0.5ml * soaking time)

Enzyme coefficient alive=0.1227 (CPC-Na is a substrate)

Dried fungus strain number=0.91 (is suitable for OD ₆₀₀Between 0.4-0.8)

Embodiment 7 former engineering bacteria A _56-3/ pMR24 and new construction bacterium MMR204/pMR24 are applied to GL-7ACA comparison (data see Table 5) to the destructiveness of beta-lactam nucleus in the conversion process of 7ACA

1. the preparation of engineering bacteria: picking one single bacterium colony from the LB flat board, being inoculated in the 500ml triangle that 60ml LB solution is housed shakes in the bottle, after 37 ℃ of joltings are cultivated 12 hours, transfer with 1% inoculum size and to shake in the bottle in the 500ml triangle that 60ml LB solution is housed, cultivated 40 hours in 37 ℃ of joltings, centrifugal.

2. prepare 0.133223mg/ml GL-7ACA solution with the phosphoric acid buffer of PH7.6.

With behind engineering bacteria after centrifugal and the GL-7ACA solution mixing in 28 ℃ of water bath heat preservations 60 minutes, in this process, constantly adjust pH value with 1N NaOH, it is maintained about 7.6.

With reaction solution centrifugal after, get supernatant, with 100 times of distilled water dilutings, measure the concentration of GL-7ACA and 7ACA with high-pressure liquid phase method.

Sequence table (1) general information (I) applicant: Shanghai Inst. of Plant Physiology, Chinese Academy of Sciences's (II) denomination of invention: engineering bacteria (III) sequence number that contains glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene: 10 (2) SEQ ID NO:1 information (I) sequence signatures:

(A) length: 2482 bases

(B) type: nucleic acid

(C) chain: two strands

(D) topological structure: linear (II) molecule type: thymus nucleic acid (gene) (III) source

(A) bacterial classification: pseudomonas (Pseudomonas)

(B) bacterial strain: sp.130 (IV) feature

(A) title: signal peptide

(B) position: 104-202 or 104-223

(C) out of Memory: Glularyl-7-amino-cephalo-alkanoic acid acylase/signal peptide (IV) feature

(A) title: ripe subunit-polypeptide

(B) position: 203-667 or 224-667

(C) out of Memory: Glularyl-7-amino-cephalo-alkanoic acid acylase/Alpha-subunit (IV) feature

(A) title: space peptide-polypeptide

(B) position: 668-697

(C) out of Memory: Glularyl-7-amino-cephalo-alkanoic acid acylase/space peptide (IV) feature

(A) title: ripe subunit-polypeptide

(B) position: 698-2263

(C) out of Memory: Glularyl-7-amino-cephalo-alkanoic acid acylase/Beta-subunit (V) sequence description: SEQ ID NO:1:1 G GAT CCG TGG TTC GTA CGC GCC GCC TAC AAG TGG TGA TCT AGG GGA ACG TTC CGG GGG 58 59 CGT CGC TGC AAC GGC GCT TCC GGA TCT GGG TGA GAG GGG AAA TCC ATG CTG AGA GTT CTG 118

Met?Leu?Arg?Val?Leu

1???????????????5119??CAC?CGG?GCG?GCG?TCC?GCC?TTG?GTT?ATG?GCG?ACT?GTG?ATC?GGC?CTT?GCG?CCC?GCC?GTC?GCC??178

His?Arg?Ala?Ala?Ser?Ala?Leu?Val?Met?Ala?Thr?Val?Ile?Gly?Leu?Ala?Pro?Ala?Val?Ala

10??????????????????15??????????????????20??????????????????25179??TTT?GCC?CTG?GCC?GAG?CCG?ACC?TCG?ACG?CCG?CAG?GCG?CCG?ATT?GCG?GCC?TAT?AAA?CCG?AGA??238

Phe?Ala?Leu?Ala?Glu?Pro?Thr?Ser?Thr?Pro?Gln?Ala?Pro?Ile?Ala?Ala?Tyr?Lys?Pro?Arg

30??????????????????35??????????????????40??????????????????45239??AGC?AAT?GAG?ATC?CTG?TGG?GAC?GGC?TAC?GGC?GTC?CCG?CAC?ATC?TAC?GGC?GTC?GAC?GCG?CCC??298

Ser?Asn?Glu?Ile?Leu?Trp?Asp?Gly?Tyr?Gly?Val?Pro?His?Ile?Tyr?Gly?Val?Asp?Ala?Pro

50??????????????????55??????????????????60??????????????????65299??TCA?GCC?TTC?TAC?GGC?TAT?GGC?TGG?GCC?CAG?GCG?CGC?AGC?CAC?GGC?GAC?AAT?ATC?CTG?CGC??358

Ser?Ala?Phe?Tyr?Gly?Tyr?Gly?Trp?Ala?Gln?Ala?Arg?Ser?His?Gly?Asp?Asn?Ile?Leu?Arg

70??????????????????75??????????????????80??????????????????85359??CTG?TAT?GGA?GAA?GCG?CGG?GGC?AAG?GGG?GCC?GAA?TAC?TGG?GGC?CCG?GAT?TAC?GAA?CAG?ACG??418

Leu?Tyr?Gly?Glu?Ala?Arg?Gly?Lys?Gly?Ala?Glu?Tyr?Trp?Gly?Pro?Asp?Tyr?Glu?Gln?Thr

90??????????????????95?????????????????100?????????????????105419??ACC?GTC?TGG?CTG?CTG?ACC?AAC?GGC?GTG?CCG?GAG?CGC?GCT?CAG?CAG?TGG?TAT?GCG?CAG?CAG??478

Thr?Val?Trp?Leu?Leu?Thr?Asn?Gly?Val?Pro?Glu?Arg?Ala?Gln?Gln?Trp?Tyr?Ala?Gln?Gln

110?????????????????115?????????????????120?????????????????125479??TCG?CCT?GAT?TTC?CGC?GCC?AAC?CTC?GAC?GCC?TTC?GCG?GCG?GGC?ATC?AAC?GCC?TAT?GCG?CAG??538

Ser?Pro?Asp?Phe?Arg?Ala?Asn?Leu?Asp?Ala?Phe?Ala?Ala?Gly?Ile?Asn?Ala?Tyr?Ala?Gln

130?????????????????135?????????????????140?????????????????145539??CAG?AAC?CCC?GAC?GAC?ATC?TCG?CCC?GAC?GTG?CGG?CAG?GTG?CTG?CCG?GTT?TCC?GGC?GCC?GAC??598

Gln?Asn?Pro?Asp?Asp?Ile?Ser?Pro?Asp?Val?Arg?Gln?Val?Leu?Pro?Val?Ser?Gly?Ala?Asp

150?????????????????155?????????????????160?????????????????165599??GTG?GTG?GCC?CAC?GCC?CAC?CGC?CTG?ATG?AAC?TTC?CTC?TAT?GTC?GCG?TCG?CCC?GGC?CGC?ACC??658

Val?Val?Ala?His?Ala?His?Arg?Leu?Met?Asn?Phe?Leu?Tyr?Val?Ala?Ser?Pro?Gly?Arg?Thr

170?????????????????175?????????????????180?????????????????185659??CTG?GGC?GAG?GGC?GAC?CCG?CCG?GAC?CTG?GCC?GAT?CAA?GGA?TCC?AAC?TCC?TGG?GCG?GTG?GCG??718

Leu?Gly?Glu?Gly?Asp?Pro?Pro?Asp?Leu?Ala?Asp?Gln?Gly?Ser?Asn?Ser?Trp?Ala?Val?Ala

190?????????????????195?????????????????200?????????????????205719??CCG?GGA?AAG?ACG?GCG?AAC?GGG?AAC?GCC?CTG?CTG?CTG?CAG?AAC?CCG?CAC?CTG?TCC?TGG?ACG??778

Pro?Gly?Lys?Thr?Ala?Asn?Gly?Asn?Ala?Leu?Leu?Leu?Gln?Asn?Pro?His?Leu?Ser?Trp?Thr

210?????????????????215?????????????????220?????????????????225779??ACG?GAC?TAC?TTC?ACC?TAC?TAC?GAG?GCG?CAT?CTC?GTC?ACG?CCG?GAC?TTC?GAA?ATC?TAT?GGC??838

Thr?Asp?Tyr?Phe?Thr?Tyr?Tyr?Glu?Ala?His?Leu?Val?Thr?Pro?Asp?Phe?Glu?Ile?Tyr?Gly

230?????????????????235?????????????????240?????????????????245839??GCG?ACC?CAG?ATC?GGC?CTG?CCG?GTC?ATC?CGC?TTC?GCC?TTC?AAC?CAG?CGG?ATG?GGC?ATC?ACC??898

Ala?Thr?Gln?Ile?Gly?Leu?Pro?Val?Ile?Arg?Phe?Ala?Phe?Asn?Gln?Arg?Met?Gly?Ile?Thr

250?????????????????255?????????????????260?????????????????265899??AAT?ACC?GTC?AAC?GGC?ATG?GTG?GGG?GCC?ACC?AAC?TAT?CGG?CTG?ACG?CTT?CAG?GAC?GGC?GCC??958

Asn?Thr?Val?Asn?Gly?Met?Val?Gly?Ala?Thr?Asn?Tyr?Arg?Leu?Thr?Leu?Gln?Asp?Gly?Gly

270?????????????????275?????????????????280?????????????????285959??TAT?CTG?TAT?GAC?GGT?CAG?GTG?CGG?CCG?TTC?GAG?CGG?CGT?CAG?GCC?TCG?TAT?CGC?CTG?CGT?1018

Tyr?Leu?Tyr?Asp?Gly?Gln?Val?Arg?Pro?Phe?Glu?Arg?Arg?Gln?Ala?Ser?Tyr?Arg?Leu?Arg

290?????????????????295??????????????????300??????????????????3051019??CAG?GCG?GAC?GGG?ACG?ACG?GTC?GAC?AAG?CCG?TTG?GAG?ATC?CGC?TCC?AGC?GTC?CAT?GGC?CCG???1078

Gln?Ala?Asp?Gly?Thr?Thr?Val?Asp?Lys?Pro?Leu?Glu?Ile?Arg?Ser?Ser?Val?His?Gly?Pro

310?????????????????315?????????????????320?????????????????3251079??GTC?TTC?GAG?CGC?GCG?GAC?GGC?ACG?GCC?GTC?GCC?GTT?CGG?GTC?GCC?GGT?CTG?GAC?CGG?CCG???1138

Val?Phe?Glu?Arg?Ala?Asp?Gly?Thr?Ala?Val?Ala?Val?Arg?Val?Ala?Gly?Leu?Asp?Arg?Pro

330?????????????????335?????????????????340?????????????????3451139??GGC?ATG?CTC?GAG?CAG?TAT?TTC?GAC?ATG?ATC?ACG?GCG?GAC?AGC?TTC?GAC?GAC?TAC?GAA?GCC???1198

Gly?Met?Leu?Glu?Gln?Tyr?Phe?Asp?Met?Ile?Thr?Ala?Asp?Ser?Phe?Asp?Asp?Tyr?Glu?Ala

350?????????????????355?????????????????360?????????????????3651199??GCT?TTG?GCG?CGG?ATG?CAG?GTG?CCG?ACC?TTC?AAC?ATC?GTC?TAC?GCC?GAC?CGC?GAA?GC4?ACC???1258

Ala?Leu?Ala?Arg?Met?Gln?Val?Pro?Thr?Phe?Asn?Ile?Val?Tyr?Ala?Asp?Arg?Glu?Gly?Thr

370?????????????????375?????????????????380?????????????????3851259??ATC?AAC?TAC?AGC?TTC?AAC?GGC?GTG?GCG?CCC?AAA?CGG?GCC?GAG?CGC?GAC?ATC?GCC?TTC?TGG???1318

Ile?Asn?Tyr?Ser?Phe?Asn?Gly?Val?Ala?Pro?Lys?Arg?Ala?Glu?Gly?Asp?Ile?Ala?Phe?Trp

390?????????????????395?????????????????400?????????????????4051319??CAG?GGG?CTC?GTG?CCG?GGC?GAT?TCC?TCG?CGT?TAC?CTG?TGG?ACC?GAG?ACA?CAC?CCG?CTG?GAC???1378

Gln?Gly?Leu?Val?Pro?Gly?Asp?Ser?Ser?Arg?Tyr?Leu?Trp?Thr?Glu?Thr?His?Pro?Leu?Asp

410?????????????????415?????????????????420?????????????????425?1379??GAT?CTG?CCG?CCC?GTC?ACC?AAT?CCG?CCG?GGC?GGC?TTC?GTG?CAG?AAC?TCC?AAT?GAT?CCG?CCG???1438

Asp?Leu?Pro?Arg?Val?Thr?Asn?Pro?Pro?Gly?Gly?Phe?Val?Gln?Asn?Ser?Asn?Asp?Pro?Pro

430?????????????????435?????????????????440?????????????????4451439??TGG?ACG?CCG?ACC?TGG?CCC?GTC?ACC?TAC?ACG?CCC?AAG?GAC?TTC?CCC?TCC?TAT?CTG?GCG?CCC???1498

Trp?Thr?Pro?Thr?Trp?Pro?Val?Thr?Tyr?Thr?Pro?Lys?Asp?Phe?Pro?Ser?Tyr?Leu?Ala?Pro

450?????????????????455?????????????????460?????????????????4651499??CAG?ACG?CCG?CAT?TCC?CTG?CGT?GCG?CAA?CAA?AGC?GTG?CGT?CTG?ATG?TCC?GAG?AAC?GAC?GAC???1558

Gln?Thr?Pro?His?Ser?Leu?Arg?Ala?Gln?Gln?Ser?Val?Arg?Leu?Met?Ser?Glu?Asn?Asp?Asp

470?????????????????475?????????????????480?????????????????4851559??CTG?ACG?CTG?GAG?CGC?TTC?ATG?GCG?CTG?CAG?TTG?AGC?CAT?CGC?GCC?GTC?ATG?GCC?GAC?CGC???1618

Leu?Thr?Leu?Glu?Arg?Phe?Met?Ala?Leu?Gln?Leu?Ser?His?Arg?Ala?Val?Met?Ala?Asp?Arg

490?????????????????495?????????????????500?????????????????5051619??ACC?TTG?CCG?GAC?CTG?ATC?CCG?GCC?GCC?CTG?ATC?GAC?CCC?GAT?CCC?GAG?GTC?CAG?GCG?GCG???1678

Thr?Leu?Pro?Asp?Leu?Ile?Pro?Ala?Ala?Leu?Ile?Asp?Pro?Asp?Pro?Glu?Val?Gln?Ala?Ala

510?????????????????515?????????????????520?????????????????5251679??GCG?CGC?CTG?CTG?GCG?GCG?TGG?GAT?CGC?GAG?TTC?ACC?AGC?GAC?AGC?CGC?GCC?GCC?CTG?CTG???1738

Ala?Arg?Leu?Leu?Ala?Ala?Trp?Asp?Arg?Glu?Phe?Thr?Ser?Asp?Ser?Arg?Ala?Ala?Leu?Leu

530?????????????????535?????????????????540?????????????????5451739??TTC?GAG?GAA?TGG?GCG?CGT?CTG?TTC?GCC?GGC?CAG?AAT?TTC?GCA?CGC?CAG?GCC?GGC?TTC?GCC???1798

Phe?Glu?Glu?Trp?Ala?Arg?Leu?Phe?Ala?Gly?Gln?Asn?Phe?Ala?Gly?Gln?Ala?Gly?Phe?Ala

550?????????????????555?????????????????560?????????????????5651799??ACG?CCC?TGG?TCG?CTG?GAT?AAG?CCG?GTC?AGC?ACG?CCT?TAC?GGC?GTC?CGC?GAC?CCC?AAG?GCC???1858

Thr?Pro?Trp?Ser?Leu?Asp?Lys?Pro?Val?Ser?Thr?Pro?Tyr?Gly?Val?Arg?Asp?Pro?Lys?Ala

570?????????????????575?????????????????580?????????????????5851859??GCC?GTC?GAT?CAA?CTG?CGG?ACC?GCC?ATC?GCC?AAC?ACC?AAG?CGC?AAA?TAC?GGC?GCG?ATC?GAC???1918

Ala?Val?Asp?Gln?Leu?Arg?Thr?Ala?Ile?Ala?Asn?Thr?Lys?Arg?Lys?Tyr?Gly?Ala?Ile?Asp

590?????????????????595?????????????????600?????????????????6051919??CGG?CCG?TTC?GGC?GAC?GCC?TCG?CGC?ATG?ATC?CTG?AAC?GAC?GTG?AAT?GTT?CCG?GGC?GCC?GCC???1978

Arg?Pro?Phe?Gly?Asp?Ala?Ser?Arg?Met?Ile?Leu?Asn?Asp?Val?Asn?Val?Pro?Gly?Ala?Ala

610?????????????????615?????????????????620?????????????????6251979??GGC?TAC?GGC?AAC?CTG?GGT?TCC?TTC?CGG?GTC?TTC?ACC?TGG?TCC?GAT?CCT?GAC?GAA?AAC?GGG???2038

Gly?Tyr?Gly?Asn?Leu?Gly?Ser?Phe?Arg?Val?Phe?Thr?Trp?Ser?Asp?Pro?Asp?Glu?Asn?Gly

630?????????????????635?????????????????640?????????????????6452039??GTT?CGC?ACG?CCC?GTC?CAC?GGC?GAG?ACG?TGG?GTG?GCG?ATG?ATC?GAG?TTC?TCC?ACG?CCG?GTG???2098

Val?Arg?Thr?Pro?Val?His?Gly?Glu?Thr?Trp?Val?Ala?Met?Ile?Glu?Phe?Ser?Thr?Pro?Val

650?????????????????655?????????????????660?????????????????6652099??CGG?GCC?TAT?GGC?CTG?ATG?AGC?TAC?GGC?AAC?TCT?CGC?CAG?CCG?GGC?ACG?ACG?CAC?TAC?AGC???2158

Arg?Ala?Tyr?Gly?Leu?Met?Ser?Tyr?Gly?Asn?Ser?Arg?Gln?Pro?Gly?Thr?Thr?His?Tyr?Ser

670?????????????????675?????????????????680?????????????????6852159???GAT?CAG?ATC?GAA?CGC?GTG?TCG?CGC?GCC?GAC?TTC?CGC?GAA?CTG?TTG?CTG?CGG?CGA?GAG?CAG???2218

Asp?Gln?Ile?Glu?Arg?Val?Ser?Arg?Ala?Asp?Phe?Arg?Glu?Leu?Leu?Leu?Arg?Arg?Glu?Gln

690?????????????????695?????????????????700?????????????????7052219???GTC?GAG?CCC?GCC?GTC?CAG?GAA?CGC?ACG?CCC?TTC?AAC?TTC?AAG?CCA?TGA?AAG?CCC?TGA?CCA???2278

Val?Glu?Ala?Ala?Val?Gln?Glu?Arg?Thr?Pro?Phe?Asn?Phe?Lys?Pro

710 715 7202279 TGA CAC GAC GGA TTG GGT ATT CGG CGG GCG CGG CGT CTC TGG CGC TGA TGG TCG CCG CCT, 23382339 CTG GAG CGG CGG CGG GCG AGC CGG CCT TCA CTT CGG TTC AGG TCG AGG GCT TTT CGG TTC, 23982399 CGG GCG CTC TGT CGA ACG CCT GGG CCG ACT TCG ACA ACG ACG GCG ACC TGG ACC TGG CCG, 24582459 TCT CCT GGA AGA GCG GCG AAG CTT, 2482 (3) SEQ ID NO:2 information (I) sequence signatures:

(A) length: 720 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological structure: linear (II) molecule type: protein (III) sequence description: SEQ ID NO:2:Met Leu Arg Val Leu His Arg Ala Ala Ser Ala Leu Val Met Ala Thr Val Ile Gly Leu

5??????????????????10??????????????????15??????????????????20Ala?Pro?Ala?Val?Ala?Phe?Ala?Leu?Ala?Glu?Pro?Thr?Ser?Thr?Pro?Gln?Ala?Pro?Ile?Ala

25??????????????????30??????????????????35??????????????????40Ala?Tyr?Lys?Pro?Arg?Ser?Asn?Glu?Ile?Leu?Trp?Asp?Gly?Tyr?Gly?Val?Pro?His?Ile?Tyr

45??????????????????50??????????????????55??????????????????60Gly?Val?Asp?Ala?Pro?Ser?Ala?Phe?Tyr?Gly?Tyr?Gly?Trp?Ala?Gln?Ala?Arg?Ser?His?Gly

65??????????????????70??????????????????75??????????????????80Asp?Asn?Ile?Leu?Arg?Leu?Tyr?Gly?Glu?Ala?Arg?Gly?Lys?Gly?Ala?Glu?Tyr?Trp?Gly?Pro

85??????????????????90??????????????????95?????????????????100Asp?Tyr?Glu?Gln?Thr?Thr?Val?Trp?Leu?Leu?Thr?Asn?Gly?Val?Pro?Glu?Arg?Ala?Gln?Gln

105?????????????????110?????????????????115?????????????????120Trp?Tyr?Ala?Gln?Gln?Ser?Pro?Asp?Phe?Arg?Ala?Asn?Leu?Asp?Ala?Phe?Ala?Ala?Gly?Ile

125?????????????????130?????????????????135?????????????????140Asn?Ala?Tyr?Ala?Gln?Gln?Asn?Pro?Asp?Asp?Ile?Ser?Pro?Asp?Val?Arg?Gln?Val?Leu?Pro

145?????????????????150?????????????????155?????????????????160Val?Ser?Gly?Ala?Asp?Val?Val?Ala?His?Ala?His?Arg?Leu?Met?Asn?Phe?Leu?Tyr?Val?Ala

165?????????????????170?????????????????175?????????????????180Ser?Pro?Gly?Arg?Thr?Leu?Gly?Glu?Gly?Asp?Pro?Pro?Asp?Leu?Ala?Asp?Gln?Gly?Ser?Asn

185?????????????????190?????????????????195?????????????????200Ser?Trp?Ala?Val?Ala?Pro?Gly?Lys?Thr?Ala?Asn?Gly?Asn?Ala?Leu?Leu?Leu?Gln?Asn?Pro

205?????????????????210?????????????????215?????????????????220His?Leu?Ser?Trp?Thr?Thr?Asp?Tyr?Phe?Thr?Tyr?Tyr?Glu?Ala?His?Leu?Val?Thr?Pro?Asp

225?????????????????230?????????????????235?????????????????240Phe?Glu?Ile?Tyr?Gly?Ala?Thr?Gln?Ile?Gly?Leu?Pro?Val?Ile?Arg?Phe?Ala?Phe?Asn?Gln

245?????????????????250?????????????????255?????????????????260Arg?Met?Gly?Ile?Thr?Asn?Thr?Val?Asn?Gly?Met?Val?Gly?Ala?Thr?Asn?Tyr?Arg?Leu?Thr

265?????????????????270?????????????????275?????????????????280Leu?Gln?Asp?Gly?Gly?Tyr?Leu?Tyr?Asp?Gly?Gln?Val?Arg?Pro?Phe?Glu?Arg?Arg?Gln?Ala

285?????????????????290?????????????????295?????????????????300Ser?Tyr?Arg?Leu?Arg?Gln?Ala?Asp?Gly?Thr?Thr?Val?Asp?Lys?Pro?Leu?Glu?Ile?Arg?Ser

305?????????????????310?????????????????315?????????????????320Ser?Val?His?Gly?Pro?Val?Phe?Glu?Arg?Ala?Asp?Gly?Thr?Ala?Val?Ala?Val?Arg?Val?Ala

325?????????????????330?????????????????335?????????????????340Gly?Leu?Asp?Arg?Pro?Gly?Met?Leu?Glu?Gln?Tyr?Phe?Asp?Met?Ile?Thr?Ala?Asp?Ser?Phe

345?????????????????350?????????????????355?????????????????360Asp?Asp?Tyr?Glu?Ala?Ala?Leu?Ala?Arg?Met?Gln?Val?Pro?Thr?Phe?Asn?Ile?Val?Tyr?Ala

365?????????????????370?????????????????375?????????????????380Asp?Arg?Glu?Gly?Thr?Ile?Asn?Tyr?Ser?Phe?Asn?Gly?Val?Ala?Pro?Lys?Arg?Ala?Glu?Gly

385?????????????????390?????????????????395?????????????????400Asp?Ile?Ala?Phe?Trp?Gln?Gly?Leu?Val?Pro?Gly?Asp?Ser?Ser?Arg?Tyr?Leu?Trp?Thr?Glu

405?????????????????410?????????????????415?????????????????420Thr?His?Pro?Leu?Asp?Asp?Leu?Pro?Arg?Val?Thr?Asn?ProPro?Gly?Gly?Phe?Val?Gln?Asn

425?????????????????430?????????????????435?????????????????440Ser?Asn?Asp?Pro?Pro?Trp?Thr?Pro?Thr?Trp?Pro?Val?Thr?Tyr?Thr?Pro?Lys?Asp?Phe?Pro

445?????????????????450?????????????????455?????????????????460Ser?Tyr?Leu?Ala?Pro?Gln?Thr?Pro?His?Ser?Leu?Arg?Ala?Gln?Gln?Ser?Val?Arg?Leu?Met

465?????????????????470?????????????????475?????????????????480Ser?Glu?Asn?Asp?Asp?Leu?Thr?Leu?Glu?Arg?Phe?Met?Ala?Leu?Gln?Leu?Ser?His?Arg?Ala

485?????????????????490?????????????????495?????????????????500Val?Met?Ala?Asp?Arg?Thr?Leu?Pro?Asp?Leu?Ile?Pro?Ala?Ala?Leu?Ile?Asp?Pro?Asp?Pro

505?????????????????510?????????????????515?????????????????520Glu?Val?Gln?Ala?Ala?Ala?Arg?Leu?Leu?Ala?Ala?Trp?Asp?Arg?Glu?Phe?Thr?Ser?Asp?Ser

525?????????????????530?????????????????535?????????????????540Arg?Ala?Ala?Leu?Leu?Phe?Glu?Glu?Trp?Ala?Arg?Leu?Phe?Ala?Gly?Gln?Asn?Phe?Ala?Gly

545?????????????????550?????????????????555?????????????????560Gln?Ala?Gly?Phe?Ala?Thr?Pro?Trp?Ser?Leu?Asp?Lys?Pro?Val?Ser?Thr?Pro?Tyr?Gly?Val

565?????????????????570?????????????????575?????????????????580Arg?Asp?Pro?Lys?Ala?Ala?Val?Asp?Gln?Leu?Arg?Thr?Ala?Ile?Ala?Asn?Thr?Lys?Arg?Lys

585?????????????????590?????????????????595?????????????????600Tyr?Gly?Ala?Ile?Asp?Arg?Pro?Phe?Gly?Asp?Ala?Ser?Arg?Met?Ile?Leu?Asn?Asp?Val?Asn

605?????????????????610?????????????????615?????????????????620Val?Pro?Gly?Ala?Ala?Gly?Tyr?Gly?Asn?Leu?Gly?Ser?Phe?Arg?Val?Phe?Thr?Trp?Ser?Asp

625?????????????????630?????????????????635?????????????????640Pro?Asp?Glu?Asn?Gly?Val?Arg?Thr?Pro?Val?His?Gly?Glu?Thr?Trp?Val?Ala?Met?Ile?Glu

645?????????????????650?????????????????655?????????????????660Phe?Ser?Thr?Pro?Val?Arg?Ala?Tyr?Gly?Leu?Met?Ser?Tyr?Gly?Asn?Ser?Arg?Gln?Pro?Gly

665?????????????????670?????????????????675?????????????????680Thr?Thr?His?Tyr?Ser?Asp?Gln?Ile?Glu?Arg?Val?Ser?Arg?Ala?Asp?Phe?Arg?Glu?Leu?Leu

685?????????????????690?????????????????695?????????????????700Leu?Arg?Arg?Glu?Gln?Val?Glu?Ala?Ala?Val?Gln?Glu?Arg?Thr?Pro?Phe?Asn?Phe?Lys?Pro

705 710 715 720 (4) SEQ ID NO:3 information (I) sequence signatures:

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: M13

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:3:

GTA AAA CGA CGG CCA GT (5) SEQ ID NO:4 information (I) sequence signature

(A) length: 16

(B) type: Nucleotide

(C)

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: Reverse

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:4:

AAC AGC TAT GAC CAT G (6) SEQ ID NO:5 information (I) sequence signature

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: KS

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:5:

CGA GGT CGA CGG TAT CG (7) SEQ ID NO:6 information (I) sequence signature

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: SK

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:6:

TCT AGA ACT AGT GGA TC (8) SEQ ID NO:7 information (I) sequence signature

(A) length: 18

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: ALPHA

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:7:

GCG CTG GCC GAG CCG ACC (9) SEQ ID NO:8 information (I) sequence signature

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: BETA1

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:8:

CTTCAGGACGCCGGCTA (10) SEQ ID NO:9 information (I) sequence signature

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: BETA3

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:9:

GTG CGG TCG GCC ATG AC (11) SEQ ID NO:10 information (I) sequence signature

(A) length: 17

(B) type: Nucleotide

(C) chain: strand

(D) topological structure: linear (II) molecule type: nucleic acid (III) feature:

(A) title: BETA4

(B) out of Memory: sequencing primer (IV) sequence description: SEQ ID NO:10:

GTG?AGT?TCA?CCA?GCG?AC

Claims (6)

1. the engineering bacteria MMR204/pMR24 that preservation registration number is the glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene that contains SEQ ID NO:1 of CGMCC NO.0309 is characterized in that this project bacterium is to carry the recombinant plasmid pMR24 that contains glutaryl-7-amino-cephalo phytanic acid acylated enzyme gene by engineering host bacterium MMR204 to constitute.

2. engineering bacteria MMR204/pMR24 as claimed in claim 1 is characterized in that the oligogene type that described recombinant plasmid pMR24 contains is acy, amp, and par, it is by the downstream of acy gene obtains among the plasmid pMR10 because of par recombinates with allocation base.

3. engineering bacteria MMR204/pMR24 as claimed in claim 1 is characterized in that the oligogene type that described engineering host bacterium MMR204 contains is ampC ∷ KAPA, ompT, recA.

4. the construction process of engineering bacteria MMR204/pMR24 as claimed in claim 1 is characterized in that comprising:

(1), the structure of engineering host bacterium MMR204: at first use extracorporeal recombination to obtain containing the recombinant plasmid pZJ5 of ampC ∷ KAPA sudden change, next uses recombinant technology in the body that said mutation is integrated into ampC gene locus on the escherichia coli chromosome, structure contains the bacterial strain of ampC ∷ KAPA sudden change, at last again through transforming, can make the comparatively stable proteolytic enzyme OmpT sudden change of the foreign protein that is expressed in periplasmic space and can guarantee that plasmid is stable and go down to posterity and the recA defective of clone gene stably express is introduced the host bacterium, obtain genetically engineered host bacterium MMR204;

(2), MMR204 and above-mentioned recombinant plasmid pMR24 are built into MMR204/pMR24.

5. the application of engineering bacteria MMR204/pMR24 as claimed in claim 1 is characterized in that it is the structure of the genetic engineering bacterium of purpose that described host bacterium MMR204 that this project bacterium comprises can be applicable to handle CPC and derivative thereof.

6. the application of engineering bacteria MMR204/pMR24 as claimed in claim 5 is characterized in that described CPC derivative is GL-7ACA and 7ACA.

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