CN108660122A

CN108660122A - A kind of application of transaminase, mutant and its production L-glufosinate-ammonium

Info

Publication number: CN108660122A
Application number: CN201810540980.2A
Authority: CN
Inventors: 金利群; 郑裕国; 彭凤; 程峰; 柳志强; 薛亚平; 贾东旭
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2018-10-16
Anticipated expiration: 2038-05-30
Also published as: CN108660122B

Abstract

The invention discloses a kind of transaminase, mutant and its applications for producing L glufosinate-ammoniums, and the mutant is by SEQ ID NO：The isoleucine that amino acid shown in 2 is the 62nd is substituted by valine, the 74th serine is substituted by threonine, the 93rd methionine is substituted by isoleucine, the 167th tyrosine is substituted by phenylalanine, the 220th alanine is substituted by proline, the 282nd arginine is substituted by lysine, the 353rd alanine is substituted by serine, the 355th isoleucine is substituted by valine.Transaminase prepares L glufosinate-ammoniums, product ee by substrate of 4 (methylhydroxy phosphoryl) 2 carbonyl butyric acid>99.9%, 4h substrate conversion efficiency are 56.31%.Transaminase mutant product ee>99.9%, 4h substrate conversion efficiency are 95.42%.

Description

A kind of application of transaminase, mutant and its production L-glufosinate-ammonium

(1) technical field

The invention belongs to technical field of bioengineering, are related to aminotransferase sequence Pseudomonas fluorescence (Pseudomonas Fluorescens ZJB09-108) gene, expression vector, genetic engineering bacterium and its preparing asymmetric chiral amine compound In application.

(2) background technology

Transaminase (Amine Transaminase, ATA, EC2.6.1.X) belongs to transferase, is on catalytic amino donor Transamination to the ketone compounds of latent chirality on, obtain the class of enzymes of Chiral Amine and by-product ketone or 2-ketoacid, react Process needs the participation of phosphopyridoxal pyridoxal phosphate (Pyridoxal phosphate, PLP), the reaction of catalysis to be reversible.Transaminase It is widely present in nature and plays important transamination during the nitrogen metabolism of cell.In ordinary circumstance Under, all transaminases all can according in its reversible reaction using amino acid substrate classify, according to its catalysis live Power maximum amino acid is named, such as the most suitable substrate difference of the amino group donor of aspartate transaminase and PAL enzyme For aspartic acid and phenylalanine；And be transferred on the amino acceptor of different location according to amino, transaminase can be divided again For α-transaminase and ω-transaminase.α-transaminase needs the carbonyl receptor of an alpha position to play its catalysis, but turns ammonia Enzyme in principle can be with the ketone and amine of any structure of catalyzed conversion, thus has higher application value.ω-transamination reaction mistake Journey can be divided into two steps, and first step reaction is under the action of ω-transaminase by the carbonyl of the transamination on amino group donor to PLP On, to form pyridoxamine-5-phosphate (PMP) and ketone corresponding with amino group donor；Second step reacts equally in ω-transaminase By in the transamination to amino acceptor on PMP under effect, PMP is changed into PLP and realizes cycle again.

With the continuous social and economic development, chiral aminated compounds is very important drug and pharmaceutical intermediate, Pharmaceutical industry and agricultural, chemical industry etc. play increasingly important role, and the chipal compounds of a large amount of high-purities are applied to Wherein.Currently, the preparation method of chiral aminated compounds is mainly the method for chemical synthesis and biosynthesis.Chemical synthesis is chiral Although amine compounds technique and Technical comparing are ripe, its operation is complicated, severe reaction conditions, product yield and product pair It reflects that excessive body ee values are not high, and be easy to cause the serious pollution of environment.It is compared to chemical synthesis process, biological synthesis method Remarkable advantage be that its theoretical yield can reach 100%, and reaction condition is mild, and cost is relatively low, environmental pollution It is small, the higher chiral aminated compounds of a large amount of optical purity can be made.As recombinant DNA technology and gene chemical synthesis etc. are emerging The continuous development of technology, some enzymes for being used for living things catalysis rapidly can be identified and produce, and can obtain more steady Fixed and effective biocatalyst, just because of the development of these advanced technologies, to utilize biocatalyst industrialized synthesizing chirality amine The research of class compound opens new angle.Especially recombinant DNA technology promotes the development of living things catalysis technology.Mesh Before, to be applied using enzymatic asymmetric syntheses chirality aminated compounds master is amino dehydrogenase and ω-transaminase.ω-turns Ammonia enzyme can be applied not only to the fractionation to racemic modification, can be with catalytic precursor ketone substrate asymmetric syntheses chirality amine chemical combination The optical purity of object, product is higher, and the chiral amine compound range of preparation is wider, therefore has more application value.Now ω-transaminase is found that in the wild mushroom of multiple kinds, including vibrios (Vibrio fluvialis), arthrobacterium (Arthrobacter sp.), color bacillus (Chromobacterium violaceum) etc..Koszeiewski etc. is being utilized During ATA-117 converts 4- Phenyl 2 butanones synthesis R-4- phenyl butyl- 2- amine, the DMSO extractions of integrated use organic solvent So that the conversion ratio of reactant is reached 92%, ee values with the method for adjustment pH and is up to 99%.The utilization such as Yun can co-express generation The gene recombination bacterium Escherichia coli of ω-transaminase and acetolactate synthase are catalyzed acetophenone in the form of whole cell Asymmetric syntheses phenyl ethylamine, by the way that lactic dehydrogenase is added in the reaction system, the method for removing pyruvic acid changes the heat of reaction Mechanical balance, while Product inhibiton is reduced again.Under transaminase and the collective effect of lactic dehydrogenase, 4- Phenyl 2 butanones It is converted to -4- phenyl -2- butylamine, conversion ratio reaches 99%, ee values and is more than 99%.

Glufosinate-ammonium is a kind of efficient, wide spectrum, low toxicity nonselective herbicide, is that current transgenic resistance crop is ideal Herbicide, application prospect are boundless.Glufosinate-ammonium has two kinds of enantiomters of D, L, but only L- configurations have phytotoxicity, and It easily decomposes in the soil, it is small to the destructive power of environment to the small toxicity of human and animal.Currently, glufosinate-ammonium available on the market Typically racemic mixture.If glufosinate-ammonium product can be used in the form of the optically pure isomer of L- configurations, careless ammonium can be made The usage amount of phosphine reduces by 50%, this is for improving Atom economy, reducing cost, mitigation environmental pressure all with highly important Meaning.The method for the L-glufosinate-ammonium reported at present mainly has chemical synthesis, includes fractionation, the chiral raw material of racemization glufosinate-ammonium Method, chiral auxiliary method and asymmetry catalysis method, but there are D- glufosinate-ammoniums, to be not easy racemization recycling, synthesis step tediously long, and reaction needs Ultralow temperature is wanted, the problems such as product ee values are relatively low, and yield is low, and chiral selectors are expensive.In contrast, biological synthesis process Have the advantages that stereoselectivity is stringent, reaction condition is mild and the easily separated purifying of product, therefore explores bioanalysis production L- grass The feasibility of ammonium phosphine has highly important industrial value and significant social benefit.

Natchev Ivan A are reacted using -2 ring pentanamide of L-3 amino as raw material using Michaelis-Becker, are prepared L-3- acetylaminohydroxyphenylarsonic acids 4- (hydroxymethyl phosphono) butyramide.L-3- acetylaminohydroxyphenylarsonic acids 4- (hydroxymethyl phosphono) butyramide Racemization occurs for the process that acetic acid is sloughed in acid-catalyzed hydrolysis, and product is racemization glufosinate-ammonium.Through di-phosphate ester Ι, acylase Ι and paddy After 3 enzyme stepping actions of transglutaminase, L-glufosinate-ammonium (Total synthesis and enzyme-substrate can be obtained interaction of D-,DL-,and L-phosphinotricine,‘bialaphos’(SF-1293)and its cyclic analogues[J].Journal of the Chemical Society,Perkin Transactions 1, 1989(1):125-131.), yield can reach 75.7%.This method needs the synergistic effect of 3 enzymes, and process route is longer, and And experimental cost must be increased, it is difficult to industrialize from chiral raw material.United States Patent (USP) US6686182 reports deacetylated The screening of base enzyme, purification process.At 37 DEG C, under the conditions of pH8.0, deacetylation enzymatic 48h, when concentration of substrate is less than 100mmol/L can get the glufosinate-ammonium that ee values are 100%, and as concentration of substrate increases, ee values reduce.Deacetylase is consolidated It is applied to tower reactor after fixedization, 10h is reacted using the method for current adding substrate, concentration of substrate is accumulative to reach 500mmol/L, L- grass The conversion ratio of ammonium phosphine is 83%.United States Patent (USP) US5618728 reports the method for preparing L-glufosinate-ammonium using amidase.They are sharp With Enterobacter aerogenes 9164L- amidases hydrolases (R, the S) -2- amino-screened from soil L-glufosinate-ammonium is prepared in-butyramide to 4- (hydroxymethyl phosphono), while discharging NH₃, the ee value energy of the L-glufosinate-ammonium of synthesis Reach 95.7%, the theoretical yield of amidase Split Method is 50%, and therefore, unreacted D types substrate must recycle.

In the enzymatic clarification route of many glufosinate-ammoniums, the ketone carbonyl of ketoacid intermediate is latent chiral functional group, Neng Goutong Cross enzymatic clarification approach structure chiral centre, ketone acid route also because raw material is cheap and easy to get and can to avoid using hypertoxic cyanide, And as the route suitable for L-glufosinate-ammonium industrialized developing production.

Schulz A etc. are using the transaminase being separated to from E.coli K-12, with 2- carbonyls -4- (hydroxymethyl phosphonos Amine)-butyric acid is substrate, Pidolidone is used as amino hydrogen donor, and transamination is utilized to produce L-glufosinate-ammonium (Stereospecific production of the herbicide phosphinothricin(glufosinate)by transamination: isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli[J].Applied and Environmental Microbiology,1990,56(1):1- 6.), product maximum concentration is up to 76.1g/L.Transaminase is installed after immobilization to bioreactor, the highest product of reactor Generating rate is 50g/ (L.h), and glufosinate-ammonium ee values are more than 99.9%.Chinese patent CN105603015 is reported to utilize and be derived from The transaminase of bacillus subtilis 168 (Bacillus subtis168), with 2- carbonyls -4- (hydroxymethyl phosphonic amide)-butyric acid For substrate, when concentration of substrate is 100mM, l-Alanine produces L-glufosinate-ammonium as amino hydrogen donor, using transamination, turns Rate is up to 100%.The reaction raw materials are easy to get, of low cost, environmentally protective using water phase as reaction system.But amino group donor Dosage is more, need to optimize its dosage, more economical in this way.Chinese patent CN201710178759.2 is reported in transamination reaction Ethylene synthetase is introduced, transaminase, two enzyme cascade of ethylene synthetase, at present when concentration of substrate 100mM, conversion ratio are constructed Up to 92.9%.Due to turning to introduce ethylene synthetase in ammono-system so that by-product ketone (α-ketoglutaric acid) is converted to ethylene And carbon dioxide, it removes by-product and reaction is carried out to positive reaction direction.This process simplify follow-up process for refining, improve Total recovery, but arginine and coenzyme have also been introduced simultaneously so that system is increasingly complex, is unfavorable for industrialization amplification.

Although transaminase has important application value, the rareness of transaminase enzyme gene big in preparing L-glufosinate-ammonium Its application in Chiral Amine preparation is limited greatly.Therefore, there is the novel transaminase of developmental research important theoretical research to anticipate Justice and application value.It improves the yield of L-glufosinate-ammonium and reduces cost and then transaminase method is synthesized into L-glufosinate-ammonium industrialized production It is our final goal.

(3) invention content

It is an object of the present invention to provide a kind of transaminase, mutant, the recombinant vector containing the gene, recombinant vector conversions Obtained recombination engineering bacteria, and the application in synthesis of chiral amine L-glufosinate-ammonium.For the asymmetric syntheses reported The problems such as stereoselectivity of L-glufosinate-ammonium is not high, catalyst price is high, solvent is difficult to recycle, the present invention provides a kind of catalysis The active high and strong transaminase of stereoselectivity is for catalyzing and synthesizing L-glufosinate-ammonium.

The technical solution adopted by the present invention is：

In a first aspect, the present invention, which provides one kind, deriving from Pseudomonas fluorescence (Pseudomonas fluorescens CCTCC NO:M2012539 the amino acid sequence of transaminase), the transaminase is SEQ ID NO:Shown in 2.

It is any to SEQ ID NO:The processing that amino acid is inserted into, is lacked or is replaced in amino acid sequence shown in 2 obtains Polypeptide fragment or its mutant, as long as itself and SEQ ID NO:Amino acid sequence shown in 2 has 95% or more homology, belongs to In protection scope of the present invention.

The nucleotides sequence of transaminase encoding gene of the present invention is classified as SEQ ID NO：Shown in 1, the aminotransferase gene It obtains by the following method：Using round pcr, in primer 1 (5 '-ATGTCTAAAAACGAATCTCTGCTGCAGC-3 '), primer 2 With total in Pseudomonas alba ZJB09-108 bacterial strains under the action of (5 '-TTAAGCCAGTTCGTCGAAGCATTC-3 ') Genomic DNA is that template clones aminotransferase gene sequence, is named as ata3, nucleotides sequence is classified as SEQ ID NO：Shown in 1.It should Segment is connected on pGEM-T carriers, obtains cloning vector pGEM-T-ata3, and carrier conversion Escherichia coli are obtained and contain carrier The recombination bacillus coli of pGEM-T-ata3.To recombinating plasmid order-checking, and sequencing result is analyzed using software, the sequence Containing there are one the open reading frame of long 1275bp.

3 (5 '-CCG of expression primer of the present inventionCATATGTCTAAAAACGAATCTC-3 ') and primer 4 (5 '- TTGCTCGAGTTAAGCCAGTTCGTCG-3 '), restriction enzyme site is respectively Nde I and Xho I (underscore), with cloning vector PGEM-T-ata3 is template, and the aminotransferase gene for expression has been obtained by PCR amplification.

It is any to SEQ ID NO:Nucleotide sequence shown in 1 carries out substitution, insertion or the missing of one or more nucleotide The nucleotide sequence obtained is handled, as long as it has 90% or more homology with the nucleotide, belongs to the protection of the present invention Range.

The present invention relates to containing the aminotransferase gene recombinant vector and the recombination that is converted using the recombinant vector Genetic engineering bacterium, specially：By aminotransferase gene with expression vector pET28a connections, construct containing the heterologous of aminotransferase gene Recombinant expression pET28a-ata3.Recombinant expression pET28a-ata3 is converted to E. coli BL21 (DE3) in, recombination bacillus coli E.coli BL21 (DE3)/pET28a- containing recombinant plasmid pET28a-ata3 is obtained ata3。

The invention further relates to application of the aminotransferase gene in Prepare restructuring transaminase, specially：Structure turns containing described The recombinant vector of ammonia enzyme gene converts the recombinant vector to host strain (preferably E. coli BL21 (DE3)) In, the recombination engineering bacteria of acquisition carries out Fiber differentiation, and the isolated somatic cells containing recombination transaminase of culture solution are broken The transaminase crude enzyme liquid obtained after broken is purified, and the pure enzyme of transaminase is obtained.

It is asymmetric in catalysis 2- carbonyls -4- (methylhydroxy phosphonic amide)-butyric acid (PPO) that the invention further relates to the transaminases The application in L-glufosinate-ammonium is synthesized, the specific application is：It is obtained with the fermented culture of the engineering bacteria containing aminotransferase gene wet Thalline is catalyst, and using PPO as substrate, with phosphopyridoxal pyridoxal phosphate (PLP) for coenzyme, amino group donor is added, (excellent for 6~9 in pH value Select in buffer solution 8.0) and constitute transformation system, under the conditions of 30~50 DEG C, 600rpm reaction (preferably 35 DEG C, under 600r/min Reaction is for 24 hours), the reaction was complete, and reaction solution is isolated and purified, and obtains L-glufosinate-ammonium；The amino group donor is Pidolidone, L- asparagus ferns The mixing of one or both of propylhomoserin or l-Alanine arbitrary proportion, preferably l-Alanine；In the transformation system, catalyst Dosage 20~100g/L (preferably 50g/L) is calculated as with wet thallus weight, the initial concentration of the substrate is that 10~100mM is (excellent Select 20mM), the dosage of the coenzyme is 0.1~2mM (preferably 1mM), and the amino group donor dosage is 10~80mM (preferably 20mM L-Alanine).

Further, the wet thallus that the fermented culture of the engineering bacteria of the present invention containing aminotransferase gene obtains is as follows It prepares：Engineering bacteria containing aminotransferase gene is seeded to the LB Liquid Cultures containing 50 μ g/mL kalamycin resistances of final concentration Base, 37 DEG C, cultivate 9h under 200rpm, then with 1% inoculum concentration of volumetric concentration be seeded to it is fresh containing 50 μ g/mL cards of final concentration that In the LB liquid medium of chloramphenicol resistance, cultivated to thalline OD under 37 DEG C, 150rpm₆₀₀Up to 0.6~0.8, it is added final concentration of The IPTG of 0.1mM, at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 8000rpm centrifuge 10min, discard supernatant liquid, collect wet thallus.

Second aspect, the present invention also provides a kind of transaminase mutant, the mutant is by SEQ ID NO：Ammonia shown in 2 The isoleucine of base acid the 62nd is substituted by valine, the 74th serine is substituted by threonine, the 93rd methionine Be substituted by isoleucine, the 167th tyrosine is substituted by phenylalanine, the 220th alanine is substituted by proline, 282 arginine are substituted by lysine, the 353rd alanine is substituted by serine, the 355th isoleucine is substituted by figured silk fabrics Propylhomoserin.

The nucleotides sequence of transaminase mutant code gene of the present invention is classified as SEQ ID NO：Shown in 3.

It is described the present invention also provides a kind of application of transaminase mutant in catalysis PPO synthesis L-glufosinate-ammoniums Using the wet thallus obtained using the fermented culture of the engineering bacteria of the gene of mutant code containing transaminase as catalyst, using PPO the bottom of as Amino group donor is added using phosphopyridoxal pyridoxal phosphate as coenzyme in object, the buffer solution (preferably borax-borate buffer system for being 6~9 in pH value Or sodium hydroxide-l-Alanine buffer system, pH8~9) in constitute transformation system, reacted under the conditions of 30~50 DEG C, 600rpm (preferably 35 DEG C, 600r/min under react for 24 hours), the reaction was complete, and reaction solution is isolated and purified, and obtains L-glufosinate-ammonium；The amino Donor is the mixing of one or both of Pidolidone, L-Aspartic acid or l-Alanine arbitrary proportion；The transformation system In, the dosage of catalyst is calculated as 20~100g/L (preferably 20~60g/L, more preferable 50g/L), the substrate with wet thallus weight Initial concentration be 10~500mmol/L (preferably 20~100mmol/L, more preferable 20mmol/L), the dosage of the coenzyme is 0.02~25mmol/L (preferably 0.2~2mmol/L, more preferable 1mmol/L), the amino group donor dosage be 10~ 1750mmol/L (preferably 70~350mmol/L, more preferable 80mmol/L l-Alanine).

The wet thallus that the fermented culture of engineering bacteria of the mutant gene of the present invention containing transaminase obtains is as follows It prepares：Engineering bacteria containing transaminase mutant gene is seeded to the LB liquid containing 50 μ g/mL kalamycin resistances of final concentration Body culture medium, 37 DEG C, cultivate 9h under 200rpm, then be seeded to 1% inoculum concentration of volumetric concentration and fresh contain 50 μ g/ of final concentration In the LB liquid medium of mL kalamycin resistances, cultivated to thalline OD under 37 DEG C, 150rpm₆₀₀Up to 0.6~0.8, it is added eventually The IPTG of a concentration of 0.1mM, at 28 DEG C after Fiber differentiation 12h, 4 DEG C, 8000rpm centrifuge 10min, discard supernatant liquid, collect wet Thalline.

The beneficial effects are mainly as follows：The present invention provides a kind of transaminase bases from Pseudomonas alba Aminotransferase gene is connect structure with expression vector and obtains the expression recombination containing the gene by cause, transaminase mutant gene respectively Plasmid pET28a-ata3 and pET28a-ata3-mut1, then convert into E. coli BL21 (DE3), it is recombinated Escherichia coli can carry out biocatalytic reaction as biocatalyst using recombination bacillus coli, be the biology of L-glufosinate-ammonium It catalyzes and synthesizes and provides alternative new enzyme source.

This recombination transaminase is prepared as biocatalyst by substrate of 2- carbonyls -4- (hydroxymethyl phosphono)-butyric acid L-glufosinate-ammonium, product ee>99.9%, 100mM substrate reactions for 24 hours when, conversion ratio is up to 56.31%.This recombination transaminase is prominent Variant 1 is used as biocatalyst, and L-glufosinate-ammonium, product ee are prepared as substrate using 4- (methylhydroxy phosphoryl) -2- carbonyls-butyric acid >99.9%, 100mM substrate reactions 4h, substrate conversion efficiency 95.42%；For 24 hours, substrate conversion efficiency is 500mM substrate reactions 79.71%；Compared to protoenzyme, conversion ratio improves 1.7 times or more.

(4) it illustrates

Fig. 1 is cloning vector pGEM-T-ata3 physical maps；

Fig. 2 is pET28a-ata3 recombinant plasmid physical maps；

Fig. 3 is aminotransferase gene PCR amplification low melting-point agarose gel (argrose) electrophoretogram；Wherein, swimming lane 1 is DL2000DNA Marker；Swimming lane 2 is the genetic fragment of transaminase ata3.

Fig. 4 is the SDS-PAGE figures of transaminase after purification：Swimming lane 1 is protein molecular weight Marker, and swimming lane 2 is after purification Transaminase.

Fig. 5 is that L-glufosinate-ammonium synthesizes equation.

(5) specific implementation mode

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：The amplification of aminotransferase gene ata3

Pseudomonas alba (Pseudomonas fluorescens ZJB09-108) is isolated from soil, is protected There are China typical culture collection center (deposit number CCTCC NO:M2012539 is disclosed in patent application, application Number CN 201210593105.3, open (bulletin) number CN103131649A).

It is according to the aminotransferase gene sequencing information from Pseudomonas alba (WP_076423369.1) that Genbank is included Foundation, with nucleic acid Rapid extraction instrument extraction Pseudomonas alba CCTCC NO:The total genomic dna of M2012539, with the genome DNA is template, in primer 1 (5 '-ATGTCTAAAAACGAATCTCTGCTGCAGC-3 '), primer 2 (5 '- TTAAGCCAGTTCGTCGAAGCATTC-3 ') under the action of carry out PCR amplification.PCR reaction systems (50 μ L of total volume)：10× 5 μ L, 10mM dNTP mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 1 of Pfu DNA Polymerase Buffer μ L, concentration are 50 μM of cloning primer 1, each 1 μ L of primer 2,1 μ L, Pfu DNA Polymerase of genomic DNA, 1 μ L, nothing 40 μ L of nucleic acid water.

Using the PCR instrument of BioRad, PCR reaction conditions：Pre-degeneration 95 DEG C of 5min, 95 DEG C of denaturation 30s, 65 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 recycle, last 72 DEG C of extensions 10min.

PCR reaction solution is detected with 0.9% agarose gel electrophoresis and gel extraction purifies the segment, poly- using Taq DNA Synthase is held to segment 5 ' and introduces base A.The segment is attached with pMD18-T carriers under T4DNA connection enzyme effects, is obtained Cloning recombinant plasmids pMD18-T-ata3 is shown in Fig. 1.By in the recombinant plasmid transformed to e. coli jm109, sieved using basket hickie System is selected to be screened, random picking white colonies sequencing analyzes sequencing result using software, the results showed that：Through primer 1 and draw The nucleotide sequence length that object 2 expands is 1275bp (ata3 genes, nucleotide sequence such as SEQ ID NO：Shown in 1, coding The amino acid sequence of albumen is SEQ ID NO：Shown in 2), one complete open reading frame of the sequential coding.

Embodiment 2：The structure of recombination bacillus coli E.coli BL21 (DE3)/pET28b-ata3

According to 3 (5 '-CCG of embodiment 1ata3 gene orders design primerCATATGTCTAAAAACGAATCTC-3 '), draw 4 (5 '-TTG of objectCTCGAGTTAAGCCAGTTCGTCG-3 '), and Nde I and Xho I are introduced in primer 3 and primer 4 respectively Restriction enzyme site (underscore label).Under the initiation of primer 3 and primer 4, carried out using high-fidelity Pfu archaeal dna polymerases Amplification, (obtains) in embodiment 1 by template of recombinant plasmid pMD18-T-ata3, obtains ATA3 gene orders, and Nde is utilized after sequencing I and Xho I restriction enzymes (TaKaRa) handle amplified fragments, and should using T4DNA ligases (TaKaRa) Segment with being attached with the commercial carrier pET28b (Invitrogen) of identical restriction enzyme enzymatic treatment, express by structure Carrier pET28b-ata3 (Fig. 2).The expression vector pET28b-ata3 of structure is converted to e. coli bl21 (DE3) (Invitrogen) in (42 DEG C, 90s), the LB tablets containing 50 μ g/ml kalamycin resistances is coated on, 8- is cultivated at 37 DEG C 12h, random picked clones extracting plasmid carry out sequencing identification, and screening obtains the weight containing recombinant expression pET28b-ata3 Group E. coli BL21 (DE3)/pET28b-ata3.

Embodiment 3：The induced expression of transaminase (ata3)

Recombination bacillus coli E.coli BL21 (DE3)/pET28b-ata3 that embodiment 2 obtains is seeded to containing 50 μ The LB liquid medium of g/ml kalamycin resistances, cultivates 12h under 200rpm, then be seeded to 1% (v/v) inoculum concentration by 37 DEG C In the fresh LB liquid medium containing 50 μ g/ml kalamycin resistances, in 37 DEG C, cultivated to thalline OD under 150rpm₆₀₀It reaches 0.6-0.8, is added the IPTG of final concentration of 0.1mM, at 25 DEG C after Fiber differentiation 18h, 4 DEG C, 8000rpm centrifuge 20min, discard Supernatant collects precipitation, that is, it is wet to obtain the recombination bacillus coli E.coli BL21/pET28b-ata3 containing recombinant expression Thalline.The thalline can be directly as biocatalyst.

Embodiment 4：Transaminase (ata3) whole-cell catalytic PPO prepares L-glufosinate-ammonium

Transformation system 1：In the Tris-HCl buffer systems that 10ml pH are 8.0, E.coli BL21/pET28b- are added Ata3 wet thallus 50g/L, 20mM PPO, 1mM PLP, amino group donor：L-Alanine, Pidolidone, L-Aspartic acid (20mM), It is reacted for 24 hours under the conditions of 35 DEG C, 600rpm, product ee>99.9%, substrate conversion efficiency is as shown in table 1.

Influence of the different amino group donors of table 1 to conversion ratio

Amino group donor	Conversion ratio %
		L-Aspartic acid and Pidolidone	51.54
L-Aspartic acid	58.00
		Pidolidone	54.00
L-Alanine	84.00

Transformation system 2：In the buffer system that 10ml pH are 6.0, E.coli BL21/pET28b-ata3 wet thallus is added 20g/L, 20mM PPO, 0.02mM PLP, amino group donor：L-Alanine 10mM reacts for 24 hours under the conditions of 30 DEG C, 600rpm, production Object ee>99.9%, substrate conversion efficiency 45%.

Transformation system 3：In the Tris-HCl buffer systems that 10ml pH are 8.0, E.coli BL21/pET28b- are added Ata3 wet thallus 20g/L, 20mM PPO, 1mM PLP, amino group donor：Pidolidone 80mM is anti-under the conditions of 35 DEG C, 600rpm Should for 24 hours, product ee>99.9%, substrate conversion efficiency 79%.

Transformation system 4：In the Tris-HCl buffer systems that 10ml pH are 8.0, E.coli BL21/pET28b- are added Ata3 wet thallus 30g/L, 50mM PPO, 1mM PLP, amino group donor：L-Alanine 70mM is anti-under the conditions of 35 DEG C, 600rpm Should for 24 hours, product ee>99.9%, substrate conversion efficiency 74%.

Transformation system 5：In the Tris-HCl buffer systems that 10ml pH are 9.0, E.coli BL21/pET28b- are added Ata3 wet thallus 40g/L, 60mM PPO, 1mM PLP, amino group donor：L-Alanine 40mM is anti-under the conditions of 35 DEG C, 600rpm Should for 24 hours, product ee>99.9%, substrate conversion efficiency 63.2%.

Transformation system 6：In the Tris-HCl buffer systems that 10ml pH are 9.0, E.coli BL21/pET28b- are added Ata3 wet thallus 60g/L, 100mM PPO, 2mM PLP, amino group donor：L-Alanine 40mM is anti-under the conditions of 50 DEG C, 600rpm Should for 24 hours, product ee>99.9%, substrate conversion efficiency 52%.

Embodiment 5：The foundation of ata3 gene mutation libraries

To build plasmid pET28b-ata3 in embodiment 2 as template, fallibility PCR is carried out.In primer 1 (5 '- ATGTCTAAAAACGAATCTC-3 '), fallibility PCR is carried out under the action of primer 2 (5 '-TTAAGCCAGTTCGTCGAAG-3 '). PCR reaction systems (50 μ L of total volume)：10 × Pfu DNA Polymerase Buffer, 5 μ L, 10mM dNTP mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 1 μ L, concentration are 50 μM of cloning primer 1, each 0.5 μM of primer 2, plasmid mould Plate 0.8ng/ μ L, TaqDNA Polymerase 2.5U, MnCl₂0.2mM, deionized water supply 50 μ L.Using BioRad's PCR instrument, PCR reaction conditions：Pre-degeneration 95 DEG C of 5min, 95 DEG C of denaturation 30s, 65 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 Cycle, last 72 DEG C of extensions 10min.Purify fallibility PCR product after, using the product as primer, embodiment 2 in build plasmid PET28b-ata3 is that template carries out large primer PCR, obtains large primer PCR product (i.e. mutant library 1).PCR system：Draw greatly Object 10ng/ μ L, plasmid template 1ng/ μ L, Pfu DNA Polymerase 2.5U.PCR reaction conditions：72 DEG C of 5min of A tails are removed, in advance 96 DEG C of 2min, 96 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 4min are denaturalized, totally 25 cycles, last 72 DEG C of extensions 10min。

Embodiment 6：Gene mutation library screening obtains mutant ata3-mut1

5 mutant library 1 of embodiment is transferred in the competent cell of E. coli BL21 (DE3), item is converted 42 DEG C of part, thermal shock 90 seconds, the picking monoclonal 8017 in the LB resistant panels containing 50 μ g/ml kanamycins are inoculated with respectively Induced expression is carried out in the LB culture mediums containing 50 μ g/ml kanamycins, inductive condition such as embodiment 3 obtains 8017 and contains There are the recombination bacillus coli wet thallus of mutant gene, i.e. mutant wet thallus.

After obtaining the Escherichia coli containing mutain, bioconversion, catalysis are carried out to 0.36g cremart precursor ketone PPO System (10ml) final concentration forms and catalytic condition is as follows：Mutant E.coli BL21 (DE3)/pET28b-ata3 wet thallus 0.075g, Tris-HCl buffer solution (pH 8.0), phosphopyridoxal pyridoxal phosphate 0.2mM, l-Alanine 70mM, 20mM2- carbonyl -4- (hydroxyls Methyl phosphono)-butyric acid.Reaction condition：35 DEG C of temperature, mixing speed 150r/min, the reaction time is for 24 hours.Under similarity condition, with The reaction solution that no thalline is added is replaced above-mentioned as blank control with the e. coli bl21 containing empty carrier/pET28b wet thallus Mutant wet thallus is as negative control.After reaction, sampling carries out HPLC detections (50:50 acetonitriles：Water, 10mM ammonium acetates, 0.8ml/min flow velocitys, 268nm Detection wavelengths), from 8017 mutains, there is the vigor of 6198 mutains less than original The vigor of beginning albumen, 1778 mutains is suitable with original protein, and the vigor of only 41 mutains is significantly higher than original egg (increase by 20%) in vain, the highest mutant pET28b-ata3-mut1 of conversion ratio of wherein substrate, conversion ratio 96%, ee >99%.The nucleotide sequence of mutant pET28b-ata3-mut1 and the SEQ ID No in amino acid sequence such as sequence table:3 Hes SEQ ID No:Shown in 4.Mutant 1 is by SEQ ID NO：The isoleucine that amino acid shown in 2 is the 62nd be substituted by valine, 74th serine is substituted by threonine, the 93rd methionine is substituted by isoleucine, the substitution of the 167th tyrosine For phenylalanine, the 220th alanine it is substituted by proline, the 282nd arginine is substituted by lysine, the 353rd Alanine be substituted by serine, the 355th isoleucine be substituted by valine.

Embodiment 7：Condition optimizings of transaminase mutant ata3-mut1 during turning ammonia

The recombination bacillus coli BL21/pET28b-ata3- containing recombinant expression obtained in 6 method of embodiment Mut1 wet thallus (abbreviation ata3-mut1 wet thallus) is used as biocatalyst, with 2- carbonyls -4- (hydroxymethyl phosphono)-fourth Acid is substrate.

(1) selection of amino group donor

It is constituted respectively as amino group donor using L-Aspartic acid and Pidolidone, L-Aspartic acid, Pidolidone, l-Alanine Transformation system 100mL：With pH 8.0Tris-HCl buffer solution 90mL, 20mM PPO, 24mM L-Aspartic acids, 4mM L- is added Glutamic acid, it is 50g/L to add 1mM PLP, ata3-mut1 wet thallus amounts, and 100mL is settled to Tris-HCl buffer solutions.35 DEG C, It is reacted under 600rpm for 24 hours, it is separately sampled, 10 times are diluted with ultra-pure water, HPLC detection substrate conversions are carried out using 6 method of embodiment Rate.

Under similarity condition into, amino group donor is changed to 24mM L-Aspartic acids, 80mM Pidolidones, 70mM L- third respectively Propylhomoserin the results are shown in Table shown in 2, and l-Alanine is optimal selection as amino group donor.

Influence of the different amino group donors of table 2 to conversion ratio

Amino group donor	Conversion ratio %
		L-Aspartic acid and Pidolidone	63.48
L-Aspartic acid	28.16
		Pidolidone	87.34
L-Alanine	87.21

(2) selection of buffer system

Respectively with disodium hydrogen phosphate-citrate buffer solution, phosphate buffer, Tris-HCl buffer solutions, ammonium hydroxide, borax-boron Acid buffer, sodium hydroxide are as buffer system.

Transformation system 1 (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, phosphoric acid hydrogen two Sodium-citric acid buffer system (pH 6.0-8.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 3.

3 disodium hydrogen phosphates of table-influences of the citric acid buffer system difference pH to conversion ratio

Transformation system 2 (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, phosphoric acid hydrogen two Sodium-sodium dihydrogen phosphate buffer system (pH 6.0-8.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 4.

4 disodium hydrogen phosphates of table-influences of the sodium dihydrogen phosphate buffer system difference pH to conversion ratio

Transformation system 3 (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, Tris-HCl Buffer system (pH 7.0-9.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 5.

Influences of the table 5Tris-HCl buffer system difference pH to conversion ratio

Transformation system 4 (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, ammonium hydroxide buffering System (pH7.0-9.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 6.

Influences of the 6 ammonium hydroxide buffer system difference pH of table to conversion ratio

Transformation system 5 (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, borax-boron Acid buffering system (pH 7.5-9.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 7.

7 boraxs of table-influences of the borate buffer system difference pH to conversion ratio

Transformation system 6 (10mL)：20mM PPO, 70mM l-Alanine, 1mM LPLP, wet thallus amount 50g/L, hydroxide Sodium-l-Alanine buffer system (pH 7.0-10.0), 35 DEG C, 600rpm reaction 4h, conversion results are shown in Table 8.

8 sodium hydroxides of table-influences of the l-Alanine buffer system difference pH to conversion ratio

(3) temperature optimization

Transformation system (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, wet thallus amount 50g/L, borax-boric acid Buffer system, pH 8.5 react 4h at 30 DEG C different of temperature, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 600rpm respectively, conversion Rate the results are shown in Table shown in 9.

Influence of 9 different temperatures of table to conversion ratio

(4) biomass optimizes

Transformation system (10mL)：20mM PPO, 70mM l-Alanine, 1mM PLP, borax-borate buffer system, pH 8.5, different wet thallus amount 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L are added respectively, at 40 DEG C, 600rpm 4h is reacted, conversion results are shown in Table 10.

Influence of the different biomass of table 10 to conversion ratio

Embodiment 7：The optimization of coenzyme PLP dosages

Transformation system (10mL)：20mM PPO, 70mM l-Alanine, wet thallus amount 20g/L, sodium hydroxide-l-Alanine Buffer system, pH8.5 add different PLP amounts (0.02mM-2mM), 4h, conversion results are reacted at 40 DEG C, 600rpm respectively It is shown in Table 11.

Influence of the 11 coenzyme PLP different amounts of table to conversion ratio

Embodiment 8：The optimization of amino group donor-L-Ala dosage

Transformation system (10mL)：20mM PPO, addition 1mM PLP, sodium hydroxide-l-Alanine buffer system, pH8.5, L-Alanine concentration gradient (10~80mM), wet thallus amount are 20g/L, 4h are reacted at 35 DEG C, 600rpm, conversion results are such as Shown in table 12.

Influence of the different l-Alanine dosages of table 12 to conversion ratio

Embodiment 9：Transaminase ata3-mut1 synthesizes L-glufosinate-ammonium reaction process

Transformation system (10mL)：20mM PPO, 70mM l-Alanine, is added 1mM PLP, and sodium hydroxide-l-Alanine is slow System, pH8.5 are rushed, wet thallus amount is 20g/L, and different time (1~26h), timing sampling point are reacted at 35 DEG C, 600rpm Analysis, conversion results are as shown in table 13.

Influence of the 13 differential responses time of table to conversion ratio

Embodiment 10：Reaction system (10mL)：Sodium hydroxide-l-Alanine buffer system, pH 8.5,100mM PPO, 350mM l-Alanine, addition 5mM PLP, ata3-mut1 wet thallus amounts are 20g/L, 35 DEG C, react for 24 hours under 600rpm, conversion Rate is 88.62%.

Embodiment 11：Reaction system (10mL)：Sodium hydroxide-l-Alanine buffer system, pH8.5,200mM PPO, 700mM l-Alanine, 10mM PLP, ata3-mut1 wet thallus amounts are 40g/L, 35 DEG C, react for 24 hours under 600rpm, conversion ratio It is 74.9%.

Embodiment 12：Reaction system (10mL)：Sodium hydroxide-l-Alanine buffer system, pH8.5,300mM PPO, 1050mM l-Alanine, 15mM PLP, ata3-mut1 wet thallus amounts are 60g/L, 35 DEG C, react for 24 hours under 600rpm, conversion ratio It is 85.04%.

Embodiment 13：Reaction system (10mL)：Sodium hydroxide-l-Alanine buffer system, pH8.5,400mM PPO, 1400mM l-Alanine, 20mM PLP, ata3-mut1 wet thallus amounts are 80g/L, 35 DEG C, react for 24 hours under 600rpm, conversion ratio It is 80.05%.

Embodiment 14：Reaction system (10mL)：Sodium hydroxide-l-Alanine buffer system, pH8.5,500mM PPO, 1750mM l-Alanine, 25mM PLP, ata3-mut1 wet thallus amounts are 100g/L, 35 DEG C, react for 24 hours under 600rpm, conversion Rate is 79.71%.

By the above experimental result it is found that the obtained recombination bacillus coli containing aminotransferase gene of the present invention have it is stronger Turn ammonia ability, directly can carry out living things catalysis or conversion reaction by enzyme source of the somatic cells containing enzyme, 2- carbonyls-can be utilized 4- (hydroxymethyl phosphono)-butyric acid is substrate, carries out bioconversion reaction and prepares high optically pure pesticide --- L-glufosinate-ammonium.

Sequence table

<110>Zhejiang Polytechnical University

<120>A kind of application of transaminase, mutant and its production L-glufosinate-ammonium

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1275

<212> DNA

<213>Pseudomonas fluorescence (Pseudomonas fluorescens)

<400> 1

atgtctaaaa acgaatctct gctgcagcgt cgtcaggctg ctgttgctcg tggtgtttct 60

cagatccacc cgatcgttgc tgaacgtgct gaaaacgcta ccgtttggga cgttgacggt 120

cgtgaataca tcgacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180

aaaattatcg ctgctgttca ggaacagctg accaaactga gccacacctg cttccaggtt 240

ctggcttacg aaccgtacat cgctctgtgc gaagaaatgg ctaaacgtgt tccgggtgac 300

ttcgctaaaa aaaccctgct ggttacctct ggttctgaag ctgttgaaaa cgctgttaaa 360

atcgctcgtg ctgctaccgg tcgtgctggt gttatcgctt tcaccggtgc ttaccacggt 420

cgtaccatga tgaccctgtc tctgaccggt aaagttgttc cgtactctgc tggtatgggt 480

ctgatgccgg gtggtgttta ccgtgctctg gctccgtgcc cgctgcacgg tatctctgaa 540

gacgaatcta tcgcttctat cgaacgtatc ttcaaaaacg acgctcagcc gcaggacatc 600

gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgttaa ctctaaagcg 660

ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcctgctgat cgctgacgaa 720

gttcagaccg gtgctggtcg taccggtacc ttcttcgcta ccgaacagct gggtatcgtt 780

ccggacctga ccaccttcgc taaatctgtt ggtggtggtt tcccgatctc tggtgtttgc 840

ggtagagctg aaatcatgga ctctatcgct ccgggtggtc tgggtggtac ctacgctggt 900

tctccgatcg cttgcgctgc tgctctggct gttatgaaag ttttcgacga agaaaaactg 960

ctggaacgtt ctcaggctgt tggtgaaaaa ctgaaagctg gtctgaacgc tatcgctgct 1020

aaacacaaag ttatcggtga cgttcgtggt ctgggtgcta tgattgctat cgaactgttc 1080

gaaggtggtg accacaacaa accggctgct gaactggttg gtaaaatcgt tgctcgtgct 1140

cgtgaaaaag gtctgatcct gctgtcttgc ggtacctact acaacgttat ccgtttcctg 1200

atgccggtta ccatcccgga cgctcagctg gaaaaaggta tcgctatcgt tgctgaatgc 1260

ttcgacgaac tggct 1275

<210> 2

<211> 425

<212> PRT

<213>Pseudomonas fluorescence (Pseudomonas fluorescens)

<400> 2

Met Ser Lys Asn Glu Ser Leu Leu Gln Arg Arg Gln Ala Ala Val Ala

1 5 10 15

Arg Gly Val Ser Gln Ile His Pro Ile Val Ala Glu Arg Ala Glu Asn

20 25 30

Ala Thr Val Trp Asp Val Asp Gly Arg Glu Tyr Ile Asp Phe Ala Gly

35 40 45

Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Ile Ile Ala

50 55 60

Ala Val Gln Glu Gln Leu Thr Lys Leu Ser His Thr Cys Phe Gln Val

65 70 75 80

Leu Ala Tyr Glu Pro Tyr Ile Ala Leu Cys Glu Glu Met Ala Lys Arg

85 90 95

Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Ser Gly Ser

100 105 110

Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Gly Arg

115 120 125

Ala Gly Val Ile Ala Phe Thr Gly Ala Tyr His Gly Arg Thr Met Met

130 135 140

Thr Leu Ser Leu Thr Gly Lys Val Val Pro Tyr Ser Ala Gly Met Gly

145 150 155 160

Leu Met Pro Gly Gly Val Tyr Arg Ala Leu Ala Pro Cys Pro Leu His

165 170 175

Gly Ile Ser Glu Asp Glu Ser Ile Ala Ser Ile Glu Arg Ile Phe Lys

180 185 190

Asn Asp Ala Gln Pro Gln Asp Ile Ala Ala Ile Ile Ile Glu Pro Val

195 200 205

Gln Gly Glu Gly Gly Phe Tyr Val Asn Ser Lys Ala Phe Met Gln Arg

210 215 220

Leu Arg Ala Leu Cys Asp Gln His Gly Ile Leu Leu Ile Ala Asp Glu

225 230 235 240

Val Gln Thr Gly Ala Gly Arg Thr Gly Thr Phe Phe Ala Thr Glu Gln

245 250 255

Leu Gly Ile Val Pro Asp Leu Thr Thr Phe Ala Lys Ser Val Gly Gly

260 265 270

Gly Phe Pro Ile Ser Gly Val Cys Gly Arg Ala Glu Ile Met Asp Ser

275 280 285

Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Ser Pro Ile Ala

290 295 300

Cys Ala Ala Ala Leu Ala Val Met Lys Val Phe Asp Glu Glu Lys Leu

305 310 315 320

Leu Glu Arg Ser Gln Ala Val Gly Glu Lys Leu Lys Ala Gly Leu Asn

325 330 335

Ala Ile Ala Ala Lys His Lys Val Ile Gly Asp Val Arg Gly Leu Gly

340 345 350

Ala Met Ile Ala Ile Glu Leu Phe Glu Gly Gly Asp His Asn Lys Pro

355 360 365

Ala Ala Glu Leu Val Gly Lys Ile Val Ala Arg Ala Arg Glu Lys Gly

370 375 380

Leu Ile Leu Leu Ser Cys Gly Thr Tyr Tyr Asn Val Ile Arg Phe Leu

385 390 395 400

Met Pro Val Thr Ile Pro Asp Ala Gln Leu Glu Lys Gly Ile Ala Ile

405 410 415

Val Ala Glu Cys Phe Asp Glu Leu Ala

420 425

<210> 3

<211> 1275

<212> DNA

<213>Pseudomonas fluorescence (Pseudomonas fluorescens)

<400> 3

atgtctaaaa acgaatctct gctgcagcgt cgtcaggctg ctgttgctcg tggtgtttct 60

cagatccacc cgatcgttgc tgaacgtgct gaaaacgcta ccgtttggga cgttgacggt 120

cgtgaataca tcgacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180

aaagttatcg ctgctgttca ggaacagctg accaaactga cccacacctg cttccaggtt 240

ctggcttacg aaccgtacat cgctctgtgc gaagaaatcg ctaaacgtgt tccgggtgac 300

ttcgctaaaa aaaccctgct ggttacctct ggttctgaag ctgttgaaaa cgctgttaaa 360

atcgctcgtg ctgctaccgg tcgtgctggt gttatcgctt tcaccggtgc ttaccacggt 420

cgtaccatga tgaccctgtc tctgaccggt aaagttgttc cgtactctgc tggtatgggt 480

ctgatgccgg gtggtgtttt ccgtgctctg gctccgtgcc cgctgcacgg tatctctgaa 540

gacgaatcta tcgcttctat cgaacgtatc ttcaaaaacg acgctcagcc gcaggacatc 600

gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgttaa ctctaaaccg 660

ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcctgctgat cgctgacgaa 720

gttcagaccg gtgctggtcg taccggtacc ttcttcgcta ccgaacagct gggtatcgtt 780

ccggacctga ccaccttcgc taaatctgtt ggtggtggtt tcccgatctc tggtgtttgc 840

ggtaaagctg aaatcatgga ctctatcgct ccgggtggtc tgggtggtac ctacgctggt 900

tctccgatcg cttgcgctgc tgctctggct gttatgaaag ttttcgacga agaaaaactg 960

ctggaacgtt ctcaggctgt tggtgaaaaa ctgaaagctg gtctgaacgc tatcgctgct 1020

aaacacaaag ttatcggtga cgttcgtggt ctgggttcta tggttgctat cgaactgttc 1080

gaaggtggtg accacaacaa accggctgct gaactggttg gtaaaatcgt tgctcgtgct 1140

cgtgaaaaag gtctgatcct gctgtcttgc ggtacctact acaacgttat ccgtttcctg 1200

atgccggtta ccatcccgga cgctcagctg gaaaaaggta tcgctatcgt tgctgaatgc 1260

ttcgacgaac tggct 1275

<210> 4

<211> 425

<212> PRT

<213>Pseudomonas fluorescence (Pseudomonas fluorescens)

<400> 4

Met Ser Lys Asn Glu Ser Leu Leu Gln Arg Arg Gln Ala Ala Val Ala

1 5 10 15

Arg Gly Val Ser Gln Ile His Pro Ile Val Ala Glu Arg Ala Glu Asn

20 25 30

Ala Thr Val Trp Asp Val Asp Gly Arg Glu Tyr Ile Asp Phe Ala Gly

35 40 45

Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Val Ile Ala

50 55 60

Ala Val Gln Glu Gln Leu Thr Lys Leu Thr His Thr Cys Phe Gln Val

65 70 75 80

Leu Ala Tyr Glu Pro Tyr Ile Ala Leu Cys Glu Glu Ile Ala Lys Arg

85 90 95

Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Ser Gly Ser

100 105 110

Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Gly Arg

115 120 125

Ala Gly Val Ile Ala Phe Thr Gly Ala Tyr His Gly Arg Thr Met Met

130 135 140

Thr Leu Ser Leu Thr Gly Lys Val Val Pro Tyr Ser Ala Gly Met Gly

145 150 155 160

Leu Met Pro Gly Gly Val Phe Arg Ala Leu Ala Pro Cys Pro Leu His

165 170 175

Gly Ile Ser Glu Asp Glu Ser Ile Ala Ser Ile Glu Arg Ile Phe Lys

180 185 190

Asn Asp Ala Gln Pro Gln Asp Ile Ala Ala Ile Ile Ile Glu Pro Val

195 200 205

Gln Gly Glu Gly Gly Phe Tyr Val Asn Ser Lys Pro Phe Met Gln Arg

210 215 220

Leu Arg Ala Leu Cys Asp Gln His Gly Ile Leu Leu Ile Ala Asp Glu

225 230 235 240

Val Gln Thr Gly Ala Gly Arg Thr Gly Thr Phe Phe Ala Thr Glu Gln

245 250 255

Leu Gly Ile Val Pro Asp Leu Thr Thr Phe Ala Lys Ser Val Gly Gly

260 265 270

Gly Phe Pro Ile Ser Gly Val Cys Gly Lys Ala Glu Ile Met Asp Ser

275 280 285

Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Ser Pro Ile Ala

290 295 300

Cys Ala Ala Ala Leu Ala Val Met Lys Val Phe Asp Glu Glu Lys Leu

305 310 315 320

Leu Glu Arg Ser Gln Ala Val Gly Glu Lys Leu Lys Ala Gly Leu Asn

325 330 335

Ala Ile Ala Ala Lys His Lys Val Ile Gly Asp Val Arg Gly Leu Gly

340 345 350

Ser Met Val Ala Ile Glu Leu Phe Glu Gly Gly Asp His Asn Lys Pro

355 360 365

Ala Ala Glu Leu Val Gly Lys Ile Val Ala Arg Ala Arg Glu Lys Gly

370 375 380

Leu Ile Leu Leu Ser Cys Gly Thr Tyr Tyr Asn Val Ile Arg Phe Leu

385 390 395 400

Met Pro Val Thr Ile Pro Asp Ala Gln Leu Glu Lys Gly Ile Ala Ile

405 410 415

Val Ala Glu Cys Phe Asp Glu Leu Ala

420 425

Claims

1. one kind deriving from the transaminase of Pseudomonas alba (Pseudomonas fluorescens), it is characterised in that described turn The amino acid sequence of ammonia enzyme is SEQ ID NO：Shown in 2.

2. the encoding gene of transaminase described in a kind of claim 1, it is characterised in that the encoding gene nucleotides sequence is classified as SEQ ID NO：Shown in 1.

3. a kind of recombination engineering bacteria of the encoding gene structure of transaminase described in claim 2.

4. transaminase described in a kind of claim 1 synthesizes L-glufosinate-ammonium in catalysis 2- carbonyls -4- (hydroxymethyl phosphono)-butyric acid In application.

5. application as claimed in claim 4, it is characterised in that the application is passed through with the engineering bacteria of the encoding gene containing transaminase The wet thallus that fermented and cultured obtains is catalyst, using 2- carbonyls -4- (hydroxymethyl phosphono)-butyric acid as substrate, with phosphoric acid pyrrole Aldehyde of trembling is coenzyme, and amino group donor is added, and transformation system is constituted in the buffer solution that pH value is 6~9, in 30~50 DEG C, 600rpm Under the conditions of react, the reaction was complete, and reaction solution is isolated and purified, obtain L-glufosinate-ammonium；The amino group donor be Pidolidone, L- days Winter propylhomoserin or l-Alanine；In the transformation system, the dosage of catalyst is calculated as 20~60g/L, the bottom with wet thallus weight The initial concentration of object is 20~100mM, and the dosage of the coenzyme is 0.02~2mM, and the amino group donor dosage is 10~80mM.

6. application as claimed in claim 5, it is characterised in that the wet thallus is prepared as follows：Transaminase base will be contained The engineering bacteria of cause is seeded to the LB liquid medium containing 50 μ g/mL kalamycin resistances of final concentration, 37 DEG C, cultivate under 200rpm 9h, then the fresh LB Liquid Cultures containing 50 μ g/mL kalamycin resistances of final concentration are seeded to 1% inoculum concentration of volumetric concentration In base, cultivated to thalline OD under 37 DEG C, 150rpm₆₀₀Up to 0.6~0.8, the IPTG of final concentration of 0.1mM is added, is lured at 28 DEG C After leading culture 12h, 4 DEG C, 8000rpm centrifugation 10min discard supernatant liquid, collect wet thallus.

7. transaminase mutant described in a kind of claim 1, it is characterised in that the mutant is by SEQ ID NO：Ammonia shown in 2 The isoleucine of base acid the 62nd is substituted by valine, the 74th serine is substituted by threonine, the 93rd methionine Be substituted by isoleucine, the 167th tyrosine is substituted by phenylalanine, the 220th alanine is substituted by proline, 282 arginine are substituted by lysine, the 353rd alanine is substituted by serine, the 355th isoleucine is substituted by figured silk fabrics Propylhomoserin.

8. transaminase mutant code gene described in a kind of claim 7, it is characterised in that the encoding gene nucleotide sequence For SEQ ID NO：Shown in 3.

9. transaminase mutant described in a kind of claim 7 synthesizes L- in catalysis 2- carbonyls -4- (hydroxymethyl phosphoryl)-butyric acid Application in glufosinate-ammonium.

10. application as claimed in claim 9, it is characterised in that the application is with the work of the gene of mutant code containing transaminase The wet thallus that the fermented culture of journey bacterium obtains is catalyst, using 2- carbonyls -4- (hydroxymethyl phosphoryl)-butyric acid as substrate, with Phosphopyridoxal pyridoxal phosphate is coenzyme, be added amino group donor, in pH value be 6~9 buffer solution in constitute transformation system, 30~50 DEG C, It is reacted under the conditions of 600rpm, the reaction was complete, and reaction solution is isolated and purified, and obtains L-glufosinate-ammonium；The amino group donor is L- paddy ammonia Acid, L-Aspartic acid or l-Alanine；In the transformation system, the dosage of catalyst is calculated as 20~100g/ with wet thallus weight The initial concentration of L, the substrate are 10~500mM, and the dosage of the coenzyme is 0.02~25mM, and the amino group donor dosage is 10~1750mM.