CN108893455B

CN108893455B - Transaminase mutant and application thereof in producing L-glufosinate-ammonium

Info

Publication number: CN108893455B
Application number: CN201810540686.1A
Authority: CN
Inventors: 郑裕国; 程峰; 金利群; 彭凤; 薛亚平
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: 2020-07-28
Anticipated expiration: 2038-05-30
Also published as: CN108893455A

Abstract

The invention discloses a transaminase mutant and application thereof in producing L-glufosinate-ammonium, wherein wet thalli obtained by fermentation culture of recombinant escherichia coli containing transaminase mutant coding genes is used as a biocatalyst, 2-carbonyl-4- (methyl hydroxyl phosphoryl) -butyric acid is used as a substrate, pyridoxal phosphate is used as a coenzyme, an amino donor is used as an auxiliary substrate, a reaction system is formed by using a buffer solution with pH of 6-9 as a reaction medium, and a biocatalytic reaction is carried out at the temperature of 40-50 ℃ and the stirring speed of 150-.

Description

Transaminase mutant and application thereof in producing L-glufosinate-ammonium

(I) technical field

The invention belongs to the technical field of bioengineering, and relates to a transaminase gene, an encoding enzyme, a recombinant vector containing the gene, recombinant gene engineering bacteria and a recombinase obtained by transforming the recombinant vector, and application of omega-aminotransferase in preparation of an optical homochiral pesticide L-glufosinate.

(II) background of the invention

Glufosinate, known by the name of Phosphonothricin (PPT), is generally the compound 2-amino-4- [ hydroxy (methyl) phosphono ] -butyric acid or a salt thereof with an alkaline compound. The herbicide is a non-selective herbicide with high efficiency, broad spectrum and low toxicity developed by Bayer company in the last 80 th century, is an ideal herbicide for transgenic resistant crops at present, and has a very wide application prospect.

At present, glufosinate-ammonium sold on the market is generally a racemic mixture, if the glufosinate-ammonium product can be used in the form of L-configuration optical pure isomer, the use amount of glufosinate-ammonium can be reduced by 50%, and the glufosinate-ammonium has important significance for improving atom economy, reducing cost and relieving environmental pressure.

The current methods for producing L-glufosinate-ammonium mainly comprise a chemical synthesis method, a fermentation method and a biological synthesis method.

The chemical synthesis method mainly comprises a Gabriel (Gabriel) -diethyl malonate synthesis method, a Neber (Neber) rearrangement synthesis method, an Albuzov (Arbuzov) synthesis method, a high-pressure catalytic synthesis method, a Buhler-Begers (Bucherer-Bergs) method, a Strecker method synthesis method, a low-temperature directional synthesis method, a synthesis method using L-methionine as a raw material, an asymmetric catalytic hydrogenation synthesis method, an asymmetric cyano addition method and a Michael free radical addition method.

The chiral resolution method is characterized in that the chiral resolution method is a chemical synthesis chiral resolution method, and comprises the steps of chemically synthesizing racemic D L-glufosinate-ammonium or derivatives thereof, and separating D-type isomers and L-type isomers by using a chiral resolution reagent to prepare the optically pure L-glufosinate-ammonium.

In contrast, the biological method has the advantages of strict stereoselectivity, mild reaction conditions, high yield and easy separation and purification of products, so the L-glufosinate-ammonium produced by the biological method has potential industrial value and social benefit.

The biosynthesis method mainly involves protease, deacetylase, transaminase, amidase, ester hydrolase and nitrile hydratase, the transaminase is pyridoxal phosphate (P L P) -dependent enzyme, belongs to the transferase class, and catalyzes the exchange of amino groups and keto groups, and is widely present in nature and plays an important role in transamination in the nitrogen metabolism of cells.

Transamination is a reversible reaction, and 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid (PPO) can generate L-glufosinate-ammonium by a reverse reaction under the catalysis of transaminase, U.S. Pat. No. 5,975,304 reports that Sch mu L z A et al, in the last 90 years, used a transaminase isolated from E.coli K-12, produced L-glufosinate-ammonium by catalytic transamination using 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid as a substrate and L-glutamic acid as an amino donor (FIG. 1). the transaminase was immobilized and then installed in a bioreactor, the product concentration reached 76.1 g/L, the maximum yield was 50 g/(L. h), and the ee value of L-glufosinate-ammonium exceeded 99.9%.

One of the ways to solve this problem is to use L-aspartate instead of L-glutamate, L-aspartate is transaminated to form oxaloacetate, which is unstable in aqueous solution and easily decomposed into pyruvate, thus breaking the balance of transamination reaction, in view of this, U.S. Pat. No. us6335186.b1 reports that under the above-mentioned single enzyme reaction system, an oxaloacetate transaminase is added to form a double transaminase coupling reaction system (fig. 5). however, glutamate is still required to be used in the coupling reaction, and the glutamate and ketoglutarate are in equilibrium in the reaction, and their structures are very similar to that of product L-PPT, and are difficult to be removed in separation and purification, and two impurities, oxaloacetate and L-aspartate, which need to be separated are also added.

Bartsch K et al subsequently proposed a new process (see FIG. 6) in the Chinese patent application (CN 1349561) which screened for oxaloacetate transaminase, which could specifically convert 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid by directly using L-aspartic acid as an amino donor, but the process was inefficient, with a concentration of substrate PPO of 552 mmol/L, and with almost complete consumption of L-aspartic acid, which was the starting material of 700 mmol/L, only 251.9 mmol/L of product L-PPT was produced, and at the same time pyruvate, which was the impurity pyruvate, which was about 234.5 mmol/L, was produced, and the conversion of PPO, which was the starting material, was only 52%.

Disclosure of the invention

The invention aims to provide an omega-transaminase mutant and application thereof in synthesis of L-glufosinate-ammonium, and the conversion rate of a substrate is improved.

The technical scheme adopted by the invention is as follows:

the invention provides a transaminase mutant, which is prepared by converting SEQ ID NO: 4, the histidine at position 23 is substituted by glutamine, the arginine at position 34 is substituted by proline, the glutamic acid at position 116 is substituted by valine, the alanine at position 324 is substituted by glycine, the leucine at position 397 is substituted by glutamine, and the glutamic acid at position 415 is substituted by aspartic acid.

The invention also provides a coding gene of the transaminase mutant, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1, and a vector and genetic engineering bacteria constructed by the coding gene.A transaminase mutant gene ABAT2-mut1 is connected with an expression vector pET28a to construct a heterologous expression recombinant plasmid pET28a-ABAT2-mut1 containing the transaminase mutant gene, and the expression recombinant plasmid pET28a-ABAT2-mut1 is transformed into Escherichia coli E.coli B L21 (DE3) to obtain the recombinant Escherichia coli E.coli B L21 (DE3)/pET28a-ABAT2-mut1 containing the recombinant plasmid pET28a-ABAT2-mut 1.

The invention also relates to application of the transaminase mutant in biocatalytic synthesis of L-glufosinate-ammonium, wherein wet thalli obtained by fermentation culture of recombinant escherichia coli containing transaminase mutant coding genes is used as a biocatalyst, 2-carbonyl-4- (methyl hydroxyl phosphoryl) -butyric acid (PPO) is used as a substrate, pyridoxal phosphate (P L P) is used as a coenzyme, an amino donor is used as an auxiliary substrate, 6-9 buffer solution is used as a reaction medium to form a reaction system, a biocatalytic reaction is carried out at the temperature of 30-50 ℃ (preferably 40 ℃), the stirring speed of 150 and 250r/min (preferably 180r/min), and after the reaction is finished, reaction liquid is separated and purified to obtain L-glufosinate-ammonium, and the amino donor is one or a mixture of L-glutamic acid, L-aspartic acid or L-alanine in any proportion.

Furthermore, in the reaction system, the final concentration of the used wet bacteria is 10-200 g/L (preferably 10-60 g/L, most preferably 20 g/L0), the final concentration of the added substrate is 10-500 mmol/L1 (preferably 20-100 mmol/L, most preferably 20 mmol/L), the final concentration of the added coenzyme is 0.01-20 mmol/L (preferably 0.6-2 mmol/L, most preferably 1 mmol/L), and the final concentration of the added amino donor is 10-1750 mmol/L (preferably 60-80 mmol/L, most preferably 70 mmol/L).

Further, preferably, the buffer is borax-boric acid buffer, and the pH value is 8.0.

Further, it is preferable that the amino donor is L-alanine.

Further, the wet cells were prepared by inoculating the engineered bacteria containing the gene encoding the transaminase mutant to L B liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/m L, culturing at 37 ℃ and 200rpm for 12 hours, and further inoculating to fresh cells containing kanamycin at a final concentration of 50. mu.g/m L at an inoculation amount of 1% by volumeCulturing in resistant L B liquid medium at 37 deg.C and 150rpm until the OD of the cells₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, performing induction culture at 28 ℃ for 12h, centrifuging at 4 ℃ and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

Further, the method for separating and purifying the reaction liquid comprises the following steps: (1) pretreatment: concentrating the reaction solution under reduced pressure to 10-50% of the original volume, and adjusting pH to 2-5 to obtain a pretreatment solution;

(2) performing ion exchange and adsorption on the pretreatment solution obtained in the step (1) by using a sodium type 001 × 7 cation resin, wherein the ratio of the height to the diameter of an ion exchange column is 13:1, the sampling flow rate is 1BV/h, after sampling, washing 4BV with ultrapure water, wherein the L '&gTttransfer = L' &gTtL &lTt/T &gTt-glufosinate-ammonium does not adsorb but alanine adsorbs, and collecting a water washing solution;

(3) loading the water washing liquid collected in the step (2) at the flow rate of 0.5BV/h, performing ion exchange and adsorption by using hydrogen type 001 × 7 resin, wherein the height-diameter ratio of an ion exchange column is 13:1, washing the water washing liquid for 4BV by using ultrapure water after loading, then eluting the water washing liquid by using 2 mol/L ammonia water at the flow rate of 0.5BV/h, performing ninhydrin color reaction by timing sampling, and collecting target eluent (eluent which is purple in ninhydrin reaction and is used for a period of time) to obtain an effluent containing L-glufosinate;

(4) concentrating the L-glufosinate-ammonium effluent collected in the step (3) under reduced pressure to constant weight, adding a mixed solvent and polyacrylamide, heating to dissolve L-glufosinate-ammonium, slowly stirring for 12 hours at 0 ℃ to separate L-glufosinate-ammonium crystals out, and then placing the crystals in a freeze vacuum drier for freeze drying at-80 ℃ to obtain L-glufosinate-ammonium crystals, wherein the mixed solvent is a mixture with a water/acetone volume ratio of 1:9, the volume consumption of the mixed solvent is 1000ml/g based on the weight of polyacrylamide, and the mass ratio of the polyacrylamide to the constant weight L-glufosinate-ammonium is 20-25: 1.

The principle of the ion exchange method is that when the pH of the solution is smaller than the isoelectric point of the amino acid, the amino acid is in a cation form, when the pH of the solution is larger than the isoelectric point of the amino acid, the amino acid is in an anion form, when the pH of the solution is at the isoelectric point of the amino acid, the amino acid is in a zwitter-ion form, the amino acid is separated by selecting proper pH, the pH is controlled by using ultrapure water-ammonia water, the amino acid is sequentially eluted, and effluent containing L-glufosinate-ammonium is collected.

The invention has the beneficial effects that the invention provides an expressive transaminase mutant and a gene sequence, the transaminase mutant gene can be connected with an expression vector to construct an expressive recombinant plasmid pET28a-ABAT2-mut1 containing the gene, and then the expressive recombinant plasmid is transformed into Escherichia coli B L21 (DE3) to obtain recombinant Escherichia coli, the Escherichia coli contains recombinant transaminase, the recombinant Escherichia coli can be used as a biocatalyst to carry out biocatalytic reaction, and an alternative new enzyme source is provided for the biocatalytic synthesis of L-glufosinate-ammonium.

The recombinant transaminase ABAT2 mutant 1 is used as a biocatalyst, 2-carbonyl-4- (methyl hydroxyl phosphoryl) -butyric acid is used as a substrate to prepare L-glufosinate-ammonium, the ee of the product is more than 99.9 percent, the 4h substrate conversion rate is 96.44 percent, the transaminase before mutation uses 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid as the substrate to prepare L-glufosinate-ammonium, the ee of the product is more than 99.9 percent, and the 4h substrate conversion rate is 51.15 percent, the transaminase mutant ABAT2-mit1 of the invention converts 20mM 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid at 24h with the conversion rate as high as 99.5 percent, and compared with the original strain pET28a-ABAT1, the 24h conversion rate is only 69.62 percent under the same condition, the total yield of the method of the invention is 98 percent, and the e.e. value of the product is 99 percent.

(IV) description of the drawings

FIG. 1 is a physical map of the recombinant plasmid of the cloning vector pGEM-T-ABAT 2.

FIG. 2 is a physical map of pET28a-ABAT2 recombinant plasmid.

FIG. 3 shows the electrophoresis of the transaminase gene ABAT2 on a low-melting agarose gel (agarose), in which lane 1 shows a D L2000 DNA Marker and lane 2 shows a gene fragment of the transaminase ABAT 2.

FIG. 4 is an SDS-PAGE pattern of transaminases: lane M is the protein molecular weight Marker, and lane 1 is the transaminase ABAT 2.

FIG. 5 shows the reaction equation of the transaminase catalyzing substrate 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid to synthesize L-glufosinate-ammonium under the action of amino donor L-alanine and coenzyme pyridoxal phosphate.

FIG. 6 shows the reaction equation of oxaloacetate transaminase specifically converting 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid using L-aspartic acid as an amino donor in Chinese patent CN 1349561.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in each reaction system of the examples of the present invention, the initial buffer pH was maintained throughout the reaction.

Example 1: amplification of the transaminase Gene ABAT2

Pseudomonas fluorescens ZJB09-108 is isolated from soil and stored in China center for type culture Collection (preservation number CCTCC NO: M2012539, published in the patent application, application No. CN 201210593105.3, publication No. CN 103131649A).

According to the sequencing information of transaminase gene derived from Pseudomonas (WP-076423369.1) recorded by Genbank, Pseudomonas (total genomic DNA of the cells) was extracted by a rapid nucleic acid extractor, and PCR amplification was carried out using the genomic DNA as a template under the action of primer 1 (5'-ATGAACACCAACAACGCTC-3') and primer 2 (5'-TTAAGCCTGTTTAGCTTC-3'). The PCR reaction system (total volume 50. mu. L) consisted of 10 × Pfu DNA Polymerase Buffer 5. mu. L, 10mM dNTPmix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu. L, 50. mu.M each of clone primer 1 and primer 2, 1. mu. L, genomic DNA 1. mu. L, Pfu DNA Polymerase 1. mu. L, and NUCLEIC-free acid 40. mu. L.

PCR conditions using a BioRad PCR instrument: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 65 deg.C for 45s, extension at 72 deg.C for 1min for 30 cycles, and final extension at 72 deg.C for 10 min.

The PCR reaction solution was subjected to 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel, and then a base A was introduced into the 5' end of the fragment using Taq DNA polymerase. The fragment was ligated with pMD18-T vector under the action of T4 DNA ligase to obtain the cloned recombinant plasmid pMD18-T-ABAT2 shown in FIG. 1. The recombinant plasmid is transformed into Escherichia coli JM109, a basket white spot screening system is used for screening, white clone sequencing is randomly selected, a software analysis sequencing result shows that: the length of the nucleotide sequence amplified by the primer 1 and the primer 2 is 1275bp (the nucleotide sequence of the ABAT2 gene is shown as SEQ ID NO: 3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 4), and the sequence encodes a complete open reading frame.

Example 2 construction of recombinant E.coli B L21 (DE3)/pET28B-ABAT2

Primer 3 (5' -CCG) was designed based on the sequence of ABAT2 gene in example 1CATATGAACACCAACAAC-3), primer 4 (5' -TTG)CTCGAGTTAAGCCTGTTTAGC-3'), and Nde I and Xho I restriction sites (underlined) were introduced into primer 3 and primer 4, respectively, amplification was performed using high fidelity Pfu DNA polymerase under the primer 3 and primer 4, using recombinant plasmid pMD18-T-ABAT2 as a template (obtained in example 1) to obtain ABAT2 gene sequence, after sequencing, the amplified fragment was treated with Nde I and Xho I restriction enzymes (TaKaRa), and the fragment was ligated with commercial vector pET28B (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa), to construct expression vector pET28B-ABAT2 (fig. 2), the constructed expression vector pET28B-ABAT2 was transformed into escherichia coli B L (DE3) (DE 6348) (42 ℃, 90s), plated on 50 μ g/abt 2 (fig. 2), plasmid clone DNA plasmid DNA L was extracted containing kanamycin found to contain kanamycin 638, plasmid DNA L was cloned into plasmid 12 (abva 638) and cloned into plasmid 12).

Example 3: preparation of recombinant transaminase (ABAT2) wet cells

The recombinant E.coli B L21 (DE3)/pET28a-ABAT2 cells containing the recombinant plasmid pET28a-ABAT2 obtained in example 2 were inoculated into L B liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/m L, cultured at 37 ℃ and 200rpm for 12 hours, inoculated into 1% (v/v) of fresh L B liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/ml, and cultured at 37 ℃ and 150rpm until the cell OD₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, inducing and culturing at 28 deg.C for 12h, centrifuging at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting precipitate to obtain recombinant DNA containing expressionRecombinant Escherichia coli B L21 (DE3)/pET28a-ABAT2 wet thallus can be directly used as a biocatalyst or used for protein purification.

Example 4: establishment of ABAT2 gene mutation library

Error-prone PCR was carried out using plasmid pET28b-ABAT2 constructed in example 2 as a template, and an error-prone PCR was carried out under the action of primer 1 (5'-ATGAACACCAACAACGCTC-3') and primer 2 (5'-TTAAGCCTGTTTAGCTTC-3'). PCR reaction system (total volume 50. mu. L) 10 × Pfu DNA Polymerase Buffer 5. mu. L, 10mM dNTP mix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu. L, clone primer 1 and primer 2 each at a concentration of 50. mu.M, plasmid template 0.8 ng/. mu. L Polymerase 2.5U, MnCl₂0.2mM, supplementing 50 mu L with deionized water, adopting a PCR instrument of BioRad, and adopting PCR reaction conditions of pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 45s, extension at 72 ℃ for 1min, 30 cycles altogether, finally extension at 72 ℃ for 10min, purifying an error-prone PCR product, and then carrying out large primer PCR by using the product as a primer and the plasmid pET28b-ABAT2 constructed in example 2 as a template to obtain a large primer PCR product (namely mutant library 1). the PCR system comprises a large primer of 10 ng/mu L, a plasmid template of 1 ng/mu L and Pfu DNA Polymerase 2.5 U.PCR reaction conditions of removing A tail at 72 ℃ for 5min, pre-denaturation at 96 ℃ for 2min, denaturation at 96 ℃ for 30s, annealing at 60 ℃ for 45s, extension at 72 ℃ for 4min, 25 cycles altogether, and finally extension at 72 ℃ for 10 min.

Example 5: screening gene mutation library to obtain mutant ABAT2-mut1

The gene mutation library of example 4 was transferred into competent cells of E.coli B L21 (DE3) under 42 ℃ heat shock for 90 seconds, 11287 single clones were picked from L B-resistant plates containing 50. mu.g/ml kanamycin and inoculated into L B medium containing 50. mu.g/ml kanamycin for induction expression under the conditions of example 3, to obtain 11287 recombinant E.coli wet cells containing mutant genes, i.e., mutant wet cells, of the 11287 mutant proteins, 9786 mutant proteins had lower activities than the original proteins, 1476 mutant proteins had activities comparable to the original proteins, and only 25 mutant proteins had significantly higher activities (increased by 20%) than the original proteins, of which one mutant pET28B-atal-mut1 having the highest substrate conversion rate had a conversion rate of 96% and ee > 99%

After Escherichia coli containing the mutant protein is obtained, 0.18g (10mM) of glufosinate-phosphinothricin precursor ketone is subjected to biotransformation, the final concentration composition and the catalytic conditions of a catalytic system (10ml) are as follows, 0.075g of mutant wet thalli, Tris/HCl buffer solution (pH8.0), pyridoxal phosphate 0.0248g and L-alanine 0.63g are as follows, the temperature is 35 ℃, the stirring speed is 150r/min, the reaction time is 24h, under the same conditions, the reaction liquid added by the bacteria-free body is used as a blank control, Escherichia coli B L/pET 28B wet thalli containing an empty vector is used as a negative control, after the reaction is finished, a mutant pET L C detection (50:50 acetonitrile: water, 10mM ammonium acetate, 0.8ml/min, the detection wavelength 268 nm) is selected, the mutant pET28B-AB 2-AB634 with the highest substrate conversion rate is selected from 112protein, the mutant is obtained by taking the substitution of valine for glutamic acid, the substitution of aspartic acid, the amino acid, the substitution of the amino acid, the substitution of the SEQ ID No. 1, the substitution of the amino acid, the mutant pET 35, the substitution of the mutant pET 35, the substitution of the mutant, the substitution of the amino acid, the mutant pET No. 36, the substitution of the amino acid, the.

Recombinant Escherichia coli E.coli B L21 (DE3)/pET28a-ABAT2-mut1 cells containing expression recombinant plasmid pET28a-ABAT2-mut1 were inoculated into L B liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/m L, cultured at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh L B liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/ml in an inoculum size of 1% (v/v), and cultured at 37 ℃ and 150rpm until the cells OD 150rpm₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, inducing and culturing at 28 ℃ for 12h, centrifuging at 4 ℃ and 8000rpm for 10min, discarding supernatant, collecting precipitate to obtain recombinant Escherichia coli B L21 (DE3)/pET28a-ABAT2-mut1 wet thallus containing expression recombinant plasmid.

Example 6: determination of activity of recombinant Escherichia coli containing transaminase ABAT2 and mutant ABAT2-mut1

Whole cells containing transaminase ABAT2 and mutant ABAT2-mut1 were prepared by the methods of example 3 and example 5, respectively, and used for catalyzing the substrate 2-carbonyl-4- (methylhydroxyphosphoryl) -butyric acid.

The composition and the catalytic conditions of the catalytic system are as follows, transaminase ABAT2 or mutant ABAT2-mut1 whole cells (the final concentration of the bacterial mass is 50 g/L), 2-carbonyl-4- (methyl hydroxyl phosphoryl) -butyric acid (the final concentration is 10 mmol/L), P L P (the final concentration is 1 mmol/L) and L-alanine (the final concentration is 70mM) are added into 10m L Tris/HCl buffer solution (100mM, pH8.0) to form a 10m L reaction system, the reaction is carried out for 6h at 35 ℃ and the rotation speed is 180r/min, and the conversion rate of a substrate is detected by sampling HP L C.

TABLE 1 determination of the Activity of recombinant Escherichia coli (containing the transaminase ABAT2)

Strain/plasmid	Conversion rate of substrate
		Escherichia coli B L21 (DE3)	0
Escherichia coli B L21 (DE3)/pET28a	0
		Escherichia coli B L21 (DE3)/pET28a-ABAT2	69.62％
Escherichia coli B L21 (DE3)/pET28a-ABAT2-mut1	97.11％

Example 7: condition optimization of transaminase ABAT2 mutant 1 during transamination

The recombinant Escherichia coli B L21/pET 28a-ABAT2-mut1 wet cell containing the recombinant plasmid obtained in example 5 was used as a biocatalyst, and 2-carbonyl-4- (methylhydroxyphosphoryl) -butyric acid was used as a substrate.

(1) Selection of amino donors

L-aspartic acid and L-glutamic acid, L0-aspartic acid, L1-glutamic acid and L2-alanine are taken as amino donors respectively, a transformation system is prepared by preparing Tris-HCl buffer solution 90m L3, adding 0.36g (20mM) PPO (glufosinate-ammonium), 0.3192g (24mM) L4-aspartic acid, 0.00588g (4mM) L-glutamic acid, 0.0247g P L P (final concentration is 1mM), ABAT2-mut1 wet bacteria with final concentration of 50 g/L, adjusting pH to 8, fixing volume to 100m L, respectively changing the amino donors into 0.3192g (final concentration is 24mM) L-aspartic acid, 1.176g (final concentration is 80mM) L-glutamic acid, 0.6237g (final concentration is 70mM) L-alanine, simultaneously reacting for 24h at 35 ℃ and 180rpm, respectively, sampling by 10 times of HP, carrying out dilution 2C, and detecting the transformation rate shown in Table L:

TABLE 2 Effect of amino donors on conversion

Amino donor	Conversion rate%
		L aspartic acid and L glutamic acid	63.48
L aspartic acid	28.16
		L glutamic acid	85.62
L-alanine	89.69

(2) Selection of buffer system

Disodium hydrogen phosphate-citric acid buffer solution, phosphate buffer solution, Tris/HCl buffer solution, ammonia water, borax-boric acid buffer solution and sodium hydroxide are respectively used as buffer systems.

Transformation system 1. with disodium hydrogen phosphate-citric acid as a buffer, 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM), 50 g/L wet bacteria amount of ABAT2-mut1 were added, pH was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, volume was 50m L, reaction was carried out at 35 ℃ and 180rpm for 4h, HP L C was detected, and the results of transformation rate are shown in Table 3:

TABLE 3 influence of pH on the conversion

Transformation system 2. disodium hydrogen phosphate-sodium dihydrogen phosphate were used as buffer, 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM), 50 g/L ABAT2-mut1 wet bacterial amount, pH adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, constant volume to 50m L, 35 ℃, reaction at 180rpm for 4h, HP L C detection, and the results of transformation rate are shown in Table 4:

TABLE 4 influence of pH on the conversion

Transformation system 3, Tris/HCl is used as buffer, 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM) and 50 g/L of ABAT2-mut1 wet bacteria are added, pH is adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0, the volume is 50m L, 35 ℃, 180rpm is used for reaction for 4h, HP L C is used for detection, and the result of transformation rate is shown in Table 5:

TABLE 5 influence of pH on the conversion

Transformation system 4 ammonia water-L alanine is used as buffer solution, 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM), ABAT2-mut1 wet bacterial amount is 50 g/L, pH is adjusted to 7.0, 7.5, 8.0, 8.5, 9.0, constant volume is 50m L, 35 ℃, reaction is carried out at 180rpm for 4h, HP L C is used for detection, and the result of transformation rate is shown in Table 6:

TABLE 6 influence of pH on the conversion

The transformation system 5 is prepared by using borax-boric acid as a buffer solution, adding 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM), 50 g/L wet bacteria amount of ABAT2-mut1, finally adjusting the pH to 7.5, 8.0, 8.5 and 9.0, fixing the volume to 50m L, reacting at 35 ℃ and 180rpm for 4h, and the transformation rate is shown in Table 7:

TABLE 7 influence of pH on the conversion

The transformation system 6 is characterized in that sodium hydroxide-L alanine is used as a buffer solution, 0.18g (20mM) PPO (glufosinate-ammonium), 0.322g (70mM) L-alanine, 0.0124g P L P (1mM) and 50 g/L g of ABAT2-mut1 wet bacteria are weighed, the pH values are respectively adjusted to 9.0, 9.5 and 10.0, the constant volume is 50m L, 35 ℃ and 180rpm are reacted for 4h, HP L C is used for detection, and the transformation rate is shown in Table 8:

TABLE 8 influence of pH on the conversion

(3) Temperature optimization

The transformation system is prepared by preparing borax-boric acid buffer solution, adding 0.72g (20mM) PPO (glufosinate-ammonium), 1.2474g (70mM) L-alanine, 0.0248g P L P (1mM), 50 g/L of ABAT2-mut1 wet bacteria, adjusting the pH to 8.0, and fixing the volume to 200m L, respectively reacting for 4h in a water bath kettle at 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, detecting by HP L C, wherein the transformation rate is shown in Table 9:

TABLE 9 influence of temperature on conversion

(4) Bacterial load optimization

The transformation system is prepared by preparing borax-boric acid buffer solution, adding 0.72g (20mM) PPO (glufosinate acid), 1.2474g (70mM) L-alanine, 0.0248g P L P (1mM), and ABAT2-mut1 wet thalli with the final use concentrations of 10, 20, 30, 40, 50 and 60 g/L respectively, adjusting the pH to 8.0 and fixing the volume to 200m L, weighing 0.1g, 0.2g, 0.3g, 0.4g, 0.5g and 0.6g of thalli ABAT2-mut1 in each 10m L reaction system, reacting for 4 hours in a water bath at 40 ℃ and 180rpm, detecting by HP L C, wherein the transformation rate is shown in Table 10:

TABLE 10 influence of the amount of bacteria on the conversion

Example 8 optimization of the amount of coenzyme P L P

The transformation system is prepared by blending borax-boric acid buffer solution, 0.72g (20mM) PPO (glufosinate acid), 1.2474g (70mM) L-alanine, 0.02 mM-2 mM gradient P L P (Table 11), ABAT2-mut1 wet bacterium amount is 20 g/L, pH is adjusted to 8.0, constant volume is 200m L, reaction is carried out for 4h under a water bath kettle of 180rpm at 40 ℃, HP L C is used for detection, and the transformation rate is shown in Table 11:

TABLE 11 influence of the amount of coenzyme P L P on the conversion

Amount of P L P (mM)	Conversion rate/%
		0.02	56.19
0.04	61.45
		0.06	69.37
0.08	73.16
		0.1	75.98
0.2	78.12
		0.4	79.32
0.6	81.34
		0.8	85.98
1.0	89.45
		1.5	88.13
2	88.65

Example 9 optimization of the amount of amino Donor L-alanine

The transformation system is prepared by blending borax-boric acid buffer solution, 0.72g (20mM) PPO (glufosinate acid), L-alanine with different final concentrations (shown in Table 12), 0.0248g P L P (1mM), 20 g/L of ABAT2-mut1 wet bacterial amount, adjusting pH to 8.0, fixing the volume to 200m L, reacting for 4h in a water bath kettle at 40 ℃ and 180rpm, and detecting the transformation rate by HP L C (shown in Table 12):

TABLE 12 influence of the amount of amino donor L-alanine used on the conversion

Example 10 Synthesis of the transaminase ABAT2-mut1 mutant L-Glufosinate reaction procedure

Preparing borax-boric acid buffer solution, 0.36g (20mM) PPO (glufosinate-ammonium), 0.6237g (70mM) L-alanine, 0.0247g P L P (1mM), 20 g/L of ABAT2-mut1 wet bacterial amount, adjusting the pH to 8.0, fixing the volume to 100m L, reacting at 180rpm for 1-20h, and detecting HP L C, wherein the conversion rate is shown in Table 13:

TABLE 13 Effect of time on conversion

Reaction time/h	Conversion rate/%
		1	73.53
2	75.01
		3	80.83
4	84.61
		5	87.76
6	91.31
		8	91.50
10	91.51
		12	92.47
14	93.69
		16	96.32
18	99.66
		20	99.70
22	99.73
		24	99.51
26	99.72

Example 11:

preparing borax-boric acid buffer solution, adding 0.54g (100mM) PPO (glufosinate-ammonium), 0.9355g (350mM) L-alanine, 0.03705g P L P (5mM) and ABAT2-mut1 wet bacteria amount is 20 g/L, adjusting the pH to 8.0, fixing the volume to 30m L, reacting at 40 ℃ and 180rpm for different times, and determining the conversion rate as shown in Table 14:

TABLE 14

Example 12:

adding 1.35g (250mM) PPO (glufosinate-ammonium), 2.339g (875mM) L-alanine, 0.09263g P L P (7.5mM), ABAT2-mut1 wet bacterial amount of 20 g/L, adjusting pH to 8.0, fixing the volume to 30m L, reacting at 40 ℃ and 180rpm, wherein the conversion rate is shown in Table 15:

watch 15

Reaction time/h	Conversion rate/%
		10	85.92
12	84.53
		24	85.79
48	83.48

Example 13:

preparing borax-boric acid buffer solution, adding 90g (500mM) PPO (glufosinate-ammonium), 156g (1750mM) L-alanine, 6.16g P L P (15mM) and ABAT2-mut1 wet bacteria amount of 20 g/L, adjusting pH to 8.0, fixing the volume to 1L, reacting at 40 ℃ and 180rpm, and enabling the conversion rate to reach 71.62% after 24 h.

Example 14 method for separating and extracting L-glufosinate-ammonium from the enzyme conversion solution, the specific steps are as follows:

1) the 1L enzyme-converted solution (main components 180 g/L alanine, 50 g/L L-glufosinate, inorganic salts) prepared in example 13 was concentrated under reduced pressure at 60 ℃ until the volume of the original converted solution became 500m L, and the pH was adjusted to 5 with 2 mol/L aqueous sodium hydroxide solution to obtain a pretreated solution;

2) performing ion exchange and adsorption on the pretreatment solution obtained in the step 1) by using a sodium type 001 × 7 cation resin, wherein the ratio of the height to the diameter of an ion exchange column is 13:1, the flow rate of sample loading is 1BV/h, after sample loading, washing 4BV with ultrapure water, and the L &TTT transfer = L "&gTt L &/T &gTt-glufosinate-ammonium is not adsorbed and alanine is adsorbed, and collecting a water washing solution;

3) loading the water washing liquid collected in the step 2) at the flow rate of 0.5BV/h, performing ion exchange and adsorption by using hydrogen type 001 × 7 resin, wherein the height-diameter ratio of an ion exchange column is 13:1, washing the loaded water washing liquid by using ultrapure water for 4BV, then eluting the water washing liquid by using 2 mol/L ammonia water at the flow rate of 0.5BV/h, performing ninhydrin color development reaction by timing sampling (in the reaction process, a drop of effluent is dropped on filter paper, a drop of 2% ninhydrin solution is dropped on the filter paper, drying the filter paper by blowing, if the filter paper is purple, the effluent is L-glufosinate-ammonium, and if the filter paper is not purple, the effluent is not L-glufosinate-ammonium eluate), and collecting the eluate (the eluate for a period of time, which is developed by ninhydrin reaction) to obtain the eluate containing L-glufos;

4) concentrating L-glufosinate-ammonium effluent collected in the step 3) under reduced pressure to constant weight (57.9g), adding 250ml of mixed solvent (water/acetone volume ratio is 1:9) and 2.5g of polyacrylamide, heating to dissolve L-glufosinate-ammonium, slowly stirring for 12 hours at 0 ℃ to separate out L-glufosinate-ammonium crystals, placing the crystals in a freeze vacuum drier at-80 ℃ for freeze drying to obtain L-glufosinate-ammonium crystals, taking out the crystals, quickly filling the crystals into a sealed bag, weighing 49.4g, and calculating the yield to be 98.8% and the purity to be 99.0%.

From the above experimental results, it is clear that the recombinant Escherichia coli containing transaminase gene obtained by the present invention has strong transaminase ability, and can directly perform biocatalysis or transformation reaction by using enzyme-containing bacterial cells as enzyme source, transaminase (B L21 (DE3)/pET28a-ABAT2 mutant 1) is used as transformation enzyme, and 2-carbonyl-4- (methylhydroxyphosphoryl) -butyric acid is used as substrate to perform biotransformation reaction to prepare L-glufosinate-ammonium with high optical purity.

Sequence listing

<110> Zhejiang industrial university

<120> transaminase mutant and application thereof in producing L-glufosinate-ammonium

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<170>SIPOSequenceListing 1.0

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atgaacacca acaacgctct gatgcagcgt cgtcacaacg ctgttccgcg tggtgttggt 60

cagatccagc cgatcttcgc tgaacgtgct gaaaactgcc ctgtttggga cgttgaaggt 120

cgtgaatacc tggacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180

ggtatcgttt ctgctgttga agctcagctg aaaaaactgt ctcacacctg cttccaggtt 240

ctggcttacg aaccgtacct ggctctgtgc gaacgtatga accagaaagt tccgggtgac 300

ttcgctaaaa aaaccctgct ggttaccacc ggttctgaag ctgttgtaaa cgctgttaaa 360

atcgctcgtg ctgctaccaa acgttctggt gctatcgctt tctctggtgc ttaccacggt 420

cgtacccact acaccctgtc tctgaccggt aaagttcacc cgtactctgc tggtatgggt 480

ctgatgccgg gtcacgttta ccgtgctctg tacccgtgcc cgctgcacaa catctctgac 540

gacgacgcta tcgcttctat cgaacgtatc ttcaaaaacg acgctgctcc ggaagacatc 600

gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgctgc ttctccggct 660

ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcatgctgat cgctgacgaa 720

gttcagtctg gtgctggtcg taccggtacc ctgttcgcta tggaacagat gggtgttgct 780

gctgacatca ccaccttcgc taaatctatc gcgggcggct tcccgctggc tggcgttacc 840

ggtcgtgctg acgttatgga cgctatcgct ccgggtggtc tgggtggtac ctacgctggt 900

aacccgatcg cttgcgctgc tgctctggct gttcttgaca tcttcgaaca ggaaaacctg 960

ctgcagaaag gtaacaccct gggtaacacc ctgcgtgacg gtctgatgga aatcgctgaa 1020

acccaccgtg aaatcggtga cgttcgtggt ctcggtgcta tgatcgctat cgaactgttc 1080

gaaaacggtg acccgggtaa accgaacgct gctctgaccg ctgacatcgt tacccgtgct 1140

cgtgaaaaag gtctgatcct gctgtcttgc ggtccgtact acaacatcca gcgtatcctg 1200

gttccgctga ccatcgaagc ttctcagatc cgtcagggtc tggatatcat cgctcagtgc 1260

ttcgacgaag ctaaacaggc ttaa 1284

<210>2

<211>427

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<213> Pseudomonas bacteria (Pseudomonas fluorescens)

<400>2

Met Asn Thr Asn Asn Ala Leu Met Gln Arg Arg His Asn Ala Val Pro

1 5 10 15

Arg Gly Val Gly Gln Ile Gln Pro Ile Phe Ala Glu Arg Ala Glu Asn

20 25 30

Cys Pro Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly

35 40 45

Gly Ile Ala Val Leu Asn Thr GlyHis Leu His Pro Gly Ile Val Ser

50 55 60

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

65 70 75 80

Leu Ala Tyr Glu Pro Tyr Leu Ala Leu Cys Glu Arg Met Asn Gln Lys

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Ala Ile Ala Phe Ser Gly Ala Tyr His Gly Arg Thr His Tyr

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Gln Gly Glu Gly Gly Phe Tyr Ala Ala Ser Pro Ala Phe Met Gln Arg

210 215 220

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

225 230 235 240

Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln

245 250 255

Met Gly Val Ala Ala Asp Ile Thr Thr Phe Ala Lys Ser Ile Ala Gly

260 265 270

Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Asp Val Met Asp Ala

275 280 285

Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala

290 295 300

Cys Ala Ala Ala Leu Ala Val Leu Asp Ile Phe Glu Gln Glu Asn Leu

305 310 315 320

Leu Gln Lys Gly Asn Thr Leu Gly Asn Thr Leu Arg Asp Gly Leu Met

325 330 335

Glu Ile Ala Glu Thr His Arg Glu Ile Gly Asp Val Arg Gly Leu Gly

340 345 350

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

355 360 365

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

370 375 380

Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Ile Gln Arg Ile Leu

385 390 395 400

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

405 410 415

Ile Ala Gln Cys Phe Asp Glu Ala Lys Gln Ala

420 425

<210>3

<211>1284

<212>DNA

<213> Pseudomonas bacteria (Pseudomonas fluorescens)

<400>3

atgaacacca acaacgctct gatgcagcgt cgtcacaacg ctgttccgcg tggtgttggt 60

cagatccacc cgatcttcgc tgaacgtgct gaaaactgcc gtgtttggga cgttgaaggt 120

cgtgaatacc tggacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180

ggtatcgttt ctgctgttga agctcagctg aaaaaactgt ctcacacctg cttccaggtt 240

ctggcttacg aaccgtacct ggctctgtgc gaacgtatga accagaaagt tccgggtgac 300

ttcgctaaaa aaaccctgct ggttaccacc ggttctgaag ctgttgaaaa cgctgttaaa 360

atcgctcgtg ctgctaccaa acgttctggt gctatcgctt tctctggtgc ttaccacggt 420

cgtacccact acaccctgtc tctgaccggt aaagttcacc cgtactctgc tggtatgggt 480

ctgatgccgg gtcacgttta ccgtgctctg tacccgtgcc cgctgcacaa catctctgac 540

gacgacgcta tcgcttctat cgaacgtatc ttcaaaaacg acgctgctcc ggaagacatc 600

gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgctgc ttctccggct 660

ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcatgctgat cgctgacgaa 720

gttcagtctg gtgctggtcg taccggtacc ctgttcgcta tggaacagat gggtgttgct 780

gctgacatca ccaccttcgc taaatctatc gcgggcggct tcccgctggc tggcgttacc 840

ggtcgtgctg acgttatgga cgctatcgct ccgggtggtc tgggtggtac ctacgctggt 900

aacccgatcg cttgcgctgc tgctctggct gttctggaca tcttcgaaca ggaaaacctg 960

ctgcagaaag ctaacaccct gggtaacacc ctgcgtgacg gtctgatgga aatcgctgaa 1020

acccaccgtg aaatcggtga cgttcgtggt ctgggtgcta tgatcgctat cgaactgttc 1080

gaaaacggtg acccgggtaa accgaacgct gctctgaccg ctgacatcgt tacccgtgct 1140

cgtgaaaaag gtctgatcct gctgtcttgc ggtccgtact acaacatcct gcgtatcctg 1200

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Arg Gly Val Gly Gln Ile His Pro Ile Phe AlaGlu Arg Ala Glu Asn

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Cys Arg Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly

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

50 55 60

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

65 70 75 80

Leu Ala Tyr Glu Pro Tyr Leu Ala Leu Cys Glu Arg Met Asn Gln Lys

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Ala Ile Ala Phe Ser Gly Ala Tyr His Gly Arg Thr His Tyr

130 135 140

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

145 150 155 160

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

165 170 175

Asn Ile Ser Asp Asp Asp Ala Ile Ala Ser Ile Glu Arg IlePhe Lys

180 185 190

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

195 200 205

Gln Gly Glu Gly Gly Phe Tyr Ala Ala Ser Pro Ala Phe Met Gln Arg

210 215 220

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

225 230 235 240

Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln

245 250 255

Met Gly Val Ala Ala Asp Ile Thr Thr Phe Ala Lys Ser Ile Ala Gly

260 265 270

Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Asp Val Met Asp Ala

275 280 285

Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala

290 295 300

Cys Ala Ala Ala Leu Ala Val Leu Asp Ile Phe Glu Gln Glu Asn Leu

305 310 315 320

Leu Gln Lys Ala Asn Thr Leu Gly Asn Thr Leu Arg Asp Gly Leu Met

325 330 335

Glu Ile Ala Glu Thr His Arg Glu Ile Gly Asp Val Arg Gly Leu Gly

340 345 350

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

355 360 365

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

370 375 380

Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Ile Leu Arg Ile Leu

385 390 395 400

Val Pro Leu Thr Ile Glu Ala Ser Gln Ile Arg Gln Gly Leu Glu Ile

405 410 415

Ile Ala Gln Cys Phe Asp Glu Ala Lys Gln Ala

420 425

Claims

1. A transaminase mutant, characterized in that the mutant is a mutant of SEQ ID NO: 4, the histidine at position 23 is substituted by glutamine, the arginine at position 34 is substituted by proline, the glutamic acid at position 116 is substituted by valine, the alanine at position 324 is substituted by glycine, the leucine at position 397 is substituted by glutamine, and the glutamic acid at position 415 is substituted by aspartic acid.

2. A gene encoding the transaminase mutant of claim 1, characterized in that the nucleotide sequence of the encoding gene is SEQ ID NO:1 is shown.

3. Use of the transaminase mutant of claim 1 for the biocatalytic synthesis of L-glufosinate-ammonium.

4. The method as claimed in claim 3, wherein the method comprises the steps of using wet thallus obtained by fermentation culture of recombinant Escherichia coli containing transaminase mutant coding genes as a biocatalyst, using 2-carbonyl-4- (methyl hydroxyl phosphoryl) -butyric acid as a substrate, pyridoxal phosphate as a coenzyme, using an amino donor as an auxiliary substrate, and using borax-boric acid buffer solution with pH of 8.0 as a reaction medium to form a reaction system, carrying out biocatalytic reaction at 30-50 ℃ and a stirring speed of 150-250r/min, and after the reaction is finished, separating and purifying the reaction solution to obtain L-glufosinate-ammonium, wherein the amino donor is one or a mixture of two of L-glutamic acid, L-aspartic acid and L-alanine in any proportion.

5. The use according to claim 4, wherein the reaction system comprises wet bacteria in a final concentration of 10-200 g/L, the substrate in a final concentration of 10-500 mmol/L, the coenzyme in a final concentration of 0.1-20 mmol/L, and the amino donor in a final concentration of 10-1750 mmol/L.

6. Use according to claim 4, wherein the amino donor is L-alanine.

7. The use according to claim 4, wherein the wet cells are prepared by inoculating the engineered bacteria containing the gene encoding the transaminase mutant into L B liquid medium containing kanamycin resistance at a final concentration of 50 μ g/m L, culturing at 37 ℃ and 200rpm for 12 hours, further inoculating into fresh L B liquid medium containing kanamycin resistance at a final concentration of 50 μ g/m L at an inoculation amount of 1% by volume, and culturing at 37 ℃ and 150rpm until the OD of the cells is reached₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, performing induction culture at 28 ℃ for 12h, centrifuging at 4 ℃ and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

8. The use of claim 4, wherein the reaction solution is separated and purified by the following steps: (1) pretreatment: concentrating the reaction solution under reduced pressure to 10-50% of the original volume, and adjusting pH to 2-5 to obtain a pretreatment solution;

(2) performing ion adsorption on the pretreatment solution obtained in the step (1) by using sodium type 001 × 7 cation resin, wherein the height-diameter ratio of an ion exchange column is 13:1, the sample loading flow rate is 1BV/h, washing 4BV by using ultrapure water after sample loading, and collecting water washing liquid;

(3) loading the water washing liquid collected in the step (2) at the flow rate of 0.5BV/h, performing ion adsorption by using hydrogen type 001 × 7 resin, wherein the height-diameter ratio of an ion exchange column is 13:1, washing the water washing liquid with ultrapure water for 4BV after loading, then eluting the water washing liquid with 2 mol/L ammonia water at the flow rate of 0.5BV/h, collecting target eluent according to ninhydrin color reaction, and obtaining an effluent liquid containing L-glufosinate;

(4) concentrating L-glufosinate-ammonium effluent liquid in the step (3) to constant weight under reduced pressure, adding a mixed solvent and polyacrylamide, heating to dissolve L-glufosinate-ammonium, slowly stirring for 12 hours at 0 ℃ to crystallize and separate L-glufosinate-ammonium, and then placing the crystal in a freeze vacuum drier at-80 ℃ for freeze drying to obtain L-glufosinate-ammonium crystals, wherein the mixed solvent is a mixture of water and acetone in a volume ratio of 1:9, the volume consumption of the mixed solvent is 100-1000ml/g by weight of polyacrylamide, and the mass ratio of the polyacrylamide to the constant weight L-glufosinate-ammonium is 20-25: 1.