CN109609477B

CN109609477B - Alpha-transaminase mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium

Info

Publication number: CN109609477B
Application number: CN201910032171.5A
Authority: CN
Inventors: 薛亚平; 贾东旭; 郑裕国; 刘子健; 徐海鹏; 李军良; 金利群; 柳志强; 程峰
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-01-14
Filing date: 2019-01-14
Publication date: 2022-02-18
Anticipated expiration: 2039-01-14
Also published as: CN109609477A

Abstract

The invention relates to an alpha-transaminase mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium. The mutant is obtained by carrying out single-site mutation or multi-site mutation on one or more of 52 th, 71 th, 81 th, 153 th, 168 th, 324 th, 341 th and 361 th positions of an amino acid sequence shown in SEQ ID: 1. The invention realizes the high-efficiency expression of the alpha-transaminase mutant gene with high conversion rate, and the highest enzyme activity is 808.1U/mg. The optimum reaction temperature of the alpha-transaminase mutant reaches 67 ℃ at most, and the conversion rate reaches 100% in the asymmetric synthesis of L-glufosinate-ammonium by catalyzing 600mM glufosinate-ammonium precursor ketone at the temperature, which is the highest level reported in the literature. The CkTA mutant solves the technical problems of low enzyme activity, poor substrate tolerance and low conversion rate of the existing alpha-TA, and has better application prospect.

Description

Alpha-transaminase mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium

(I) technical field

The invention relates to an alpha-transaminase mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium.

(II) background of the invention

Glufosinate-ammonium, 4- [ hydroxy (methyl) phosphonyl ] -DL-homoalanine (PPT), is a broad-spectrum, contact-killing, biocidal and non-residual herbicide, and has the characteristics of high efficiency, low toxicity, easy degradation and the like. PPT is a racemic mixture comprising two optical isomers, but only the L-form is phytotoxic. The herbicide glyphosate is withdrawn from the pesticide market, and the optically pure L-PPT is prepared to obtain unprecedented market opportunity.

The preparation of the optical pure L-PPT mainly comprises a chemical synthesis method, a chiral resolution method and an asymmetric synthesis method. The chemical synthesis method has various process steps, the price of the needed synthetic reagent is high, the cost investment is high, and part of the reagent has certain toxic action on the environment and human body, so the current green and environment-friendly requirements cannot be met. Chiral reagent is used in the process of preparing optically pure L-PPT by chiral resolution, the resolution yield is only 50 percent at most, the obtained D-PPT needs to be recycled after racemization, and the chiral resolution process is complex.

The asymmetric synthesis method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easy separation and purification and the like, and is suitable for large-scale preparation of L-PPT. Enzymes catalyzing such reactions mainly include glutamate dehydrogenase and transaminase, and the substrate is glufosinate-ammonium precursor ketone (2-carbonyl-4- (hydroxymethyl phosphoryl) -butyric acid, PPO). When the dehydrogenase is catalyzed, expensive NAD (P) H is used as a cofactor, so that the catalysis cost is too high.

Transaminase (TA for short, EC 2.6.1.X), a pyridoxal 5' -phosphate (PLP) -dependent enzyme, catalyzes the reversible transfer of an amino group from a suitable donor to a carbonyl acceptor. Depending on the position of the amino group to be transferred to the different amino acceptor positions, a-TA and ω -TA can be distinguished. alpha-TA catalyzes the transamination reaction in which the carbonyl group at the alpha position of the substrate receives an amino donor, and omega-TA catalyzes the transamination reaction in which the carbonyl group at the omega position of the substrate receives an amino donor. In principle, TA can catalyze and convert ketones and amines with a plurality of structures, so TA has higher application prospect and value.

At present, some L-PPT prepared by alpha-TA has been reported. The transaminase obtained by Schulz et al in Escherichia coli K-12 can reach a conversion of 76% in the preparation of L-PPT at a substrate concentration of 550mM after immobilization (Schulz A, Taggeselle P, Tripier D, et al, Stereospermatic process of the recombinant phosphoribosyltransferase (glufosinate) by fermentation and catalysis of a phosphoribosyltransferase from Escherichia coli, Applied & Environmental Microbiology,1990,56(1): 1.). Bartsch et al screened aspartate aminotransferase from soil and applied to the preparation of L-PPT, with aspartate as the amino donor and a substrate concentration of 40mM giving up to 75% conversion and a substrate concentration of 100mM giving up to 59% (K. Bartsch, Process for the preparation of L-phosphinothricine by enzymatic conversion with enzyme, U.S. patent (2005) 6936444.). The existing transaminase process has the problems of low enzyme activity, poor substrate tolerance and low product conversion rate.

Under the background, the invention provides the method for expressing the existing TA recombinase by the genetic engineering technology and carrying out molecular modification by the protein engineering technology, and the alpha-TA mutant catalyst with improved activity and substrate tolerance is used for preparing L-PPT by asymmetric synthesis, which has important significance for improving poor substrate tolerance and low enzyme activity of TA in the process of preparing L-PPT.

Disclosure of the invention

The invention aims to provide an alpha-transaminase mutant with excellent catalytic activity and substrate tolerance and application thereof in asymmetric synthesis of L-glufosinate-ammonium.

The technical scheme adopted by the invention is as follows:

an alpha-transaminase mutant consisting of a sequence shown as SEQ ID NO: 1, and the site of the mutation is one or more of the following: (1) 52 th bit, (2) 71 th bit, (3) 81 th bit, (4) 153 th bit, (5) 168 th bit, (6) 324 th bit, (7) 341 th bit, and (8) 361 th bit. The point mutation can be mutation of one, two, three, four, five, six, seven or eight amino acids in the above sites into tryptophan, leucine, valine, phenylalanine, serine, arginine, proline, lysine or isoleucine.

Preferably, the alpha-transaminase mutant consists of a sequence shown in SEQ ID NO: 1 is obtained by mutating one or more of the following sites: (1) the mutation of proline at position 52 to valine (P52V), (2) the mutation of aspartic acid at position 71 to leucine (D71L), (3) the mutation of alanine at position 81 to serine (a81S), (4) the mutation of threonine at position 153 to arginine (T153R), (5) the mutation of asparagine at position 168 to proline (N168P), (6) the mutation of aspartic acid at position 324 to valine (D324V), (7) the mutation of alanine at position 341 to leucine (a341L), (8) the mutation of cysteine at position 361 to isoleucine (C361I).

More preferably, said alpha-transaminase mutant consists of SEQ ID NO: 1 through the eight sites mutation, the amino acid sequence is shown as SEQ ID NO: 3, respectively.

The invention also relates to application of the alpha-transaminase mutant in catalyzing asymmetric synthesis of L-glufosinate-ammonium from glufosinate-ammonium precursor ketone. The key point of the invention lies in the selection of mutation sites, on the premise of known mutant sites, a person skilled in the art can design a mutation primer of site-directed mutation according to the TA gene (CkTA) of SEQ ID NO.2, construct a mutant by site-directed mutation by taking a cloning vector carrying the TA as a template, convert a recombinant plasmid into Escherichia coli BL21(DE3) cells or host cells capable of expressing the enzyme by taking a plasmid pET28b or a vector capable of expressing the enzyme as an expression vector, and perform fermentation culture on the positive monoclonal after high-throughput screening verification to obtain wet thalli containing the mutant.

Specifically, the application is as follows: taking wet thalli obtained by fermenting and culturing recombinant genetic engineering bacteria containing the alpha-transaminase mutant coding gene or supernatant obtained by carrying out ultrasonic disruption on the wet thalli as a catalyst, taking glufosinate-ammonium precursor ketone PPO as a substrate, taking pyridoxal phosphate as a coenzyme, taking natural amino acid as an amino donor, reacting in a Tris-HCl buffer solution with the pH value of 8.0 at the temperature of 32-77 ℃ at the speed of 400-600 r/min, and after the reaction is finished, separating and purifying reaction liquid to obtain the L-glufosinate-ammonium.

The sequence of the alpha-transaminase mutant coding gene is shown in SEQ ID NO. 4.

In the reaction system, the initial concentration of the substrate is 20 to 800mM (preferably 600mM), the amount of wet cells is 10 to 100g/L (preferably 50g/L), and the amount of coenzyme is 0 to 1mM (preferably 0.2 mM).

Preferably, the reaction is carried out at 57 to 77 ℃ (preferably 67 ℃).

Specifically, the wet cells can be prepared as follows: constructing a recombinant vector containing the alpha-TA mutant gene with excellent catalytic activity and substrate tolerance, transforming the recombinant vector into E.coli, performing induced expression on the obtained recombinant gene engineering bacteria, and separating culture solution to obtain wet bacterial cells. The method specifically comprises the following steps: inoculating the engineering bacteria containing the alpha-TA mutant gene to an LB liquid culture medium containing 50 mu g/mL kanamycin, and culturing at 37 ℃ and 150r/min for 10h to obtain a seed solution; inoculating the seed solution into a fresh LB culture medium containing 50 mug/mL kanamycin at a final concentration by 2% (v/v), culturing OD600 to 0.6-0.8 at 37 ℃ and 150r/min, adding IPTG at a final concentration of 1mM into the culture solution, inducing for 12h at 28 ℃, centrifuging for 10min at 4 ℃ and 8000r/min, discarding the supernatant, and collecting wet thalli; the LB medium composition: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl and water as a solvent, and the pH value is natural.

The invention has the following beneficial effects: the invention provides a novel alpha-TA mutant, which has higher catalytic activity (up to 808.1U/mg) on glufosinate-ammonium precursor ketone PPO, has the optimum reaction temperature of 67 ℃, and has the characteristics of the maximum level reported in the literature. The mutant is used for asymmetrically producing L-PPT, the conversion rate can reach 100 percent at most, and the mutant is the highest level reported in the literature. The CkTA mutant solves the technical problems of low enzyme activity, poor substrate tolerance and low conversion rate of the existing alpha-TA, and has better application prospect.

(IV) description of the drawings

FIG. 1 is a schematic diagram of the optimum temperature of a CkTA mutant;

FIG. 2 is a schematic representation of amino donor screening of CkTA mutants;

(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:

example 1: construction and screening of CkTA single-site mutant

1. Mutant construction

Designing a mutation primer of site-directed mutation according to a CkTA parent (parent CkTA transaminase is derived from Citrobacter koseri, GenBank numbering is WP _071257673.1) (the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2), introducing single mutation to the 52 th site by using a recombinant vector pET28b/CkTA as a template by using a rapid PCR technology, wherein the primer is as follows:

forward primer CCTGTATCTGNNKATGGAAG (base mutation underlined)

Reverse primer CCTTCCTCCATNNKCAGATACAG (base mutation underlined)

And (3) PCR reaction system: 2X Phanta Max Buffer (containing Mg)²⁺)25 μ L of dNTPs 10mM, 2 μ L of forward primer, 2 μ L of reverse primer, 1 μ L of template DNA, 50U of Phanta Max Super-Fidelity DNA Polymerase, and ddH₂O to 50. mu.L.

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 59 ℃ for 6.5min) for 30 cycles; 5min at 72 ℃.

Adding 5 mu L of PCR product into 100 mu L of ice bath competent cell suspension, standing on ice for 30min, thermally shocking the transformation product at 42 ℃ for 90s, rapidly placing on ice for cooling for 2min, adding 600 mu L of LB liquid culture medium into the tube, culturing at 37 ℃ and 150r/min for 60min, coating 100 mu L of the bacterial liquid on a plate, and performing inverted culture at 37 ℃ for 12h after the bacterial liquid is completely absorbed by the culture medium.

2. High throughput screening for positive transformants

The final concentration of the prepared reaction mixed solution A consists of: 100mM Tris-HCl buffer (pH8.0), 0.1mM PLP, 20mM PPO, 60mM L-glutamic acid. The preparation of the reaction mixed solution B comprises the following steps: bromothymol blue indicator.

Picking mutant strains of the transformation plate by using toothpicks to a 96-well plate with 1mL of fermentation culture medium (LB culture medium comprises 50 mug/mL kanamycin and 1mM IPTG) in each well, sealing the edge by using a sealing film, culturing for about 10 hours at 28 ℃ and 150r/min, taking 20 muL of fermentation liquor obtained by culturing to a clean 96-well plate, adding 100 muL of A liquid preserved at 37 ℃, reacting for 30 minutes at 37 ℃ and 150r/min, adding 30 muL of B liquid to observe color change, and taking the mutant strain with the most obvious color change as a control to carry out subsequent enzyme activity determination on the mutant strain with the wild type transaminase generation strain E.coli BL21(DE3)/pET28B/CkTA (E.coli BL21(DE3) which is shown in SEQ ID NO. 2).

3. Positive transformant fermentation enzyme production

Single positive transformants were inoculated into 5mL LB containing 50. mu.g/mL kanamycinShaking culturing in test tube with culture medium at 37 deg.C and 150r/min to OD₆₀₀About 0.6-0.8, IPTG was added to the culture to a final concentration of 1mM, induction culture was carried out overnight for 12 hours at 28 ℃ and the cells were collected by centrifugation in a 1.5mL centrifuge tube.

4. Enzyme activity assay

And (2) carrying out ultrasonic disruption on the wet thalli by adopting an ultrasonic disruption method, taking 1g of the positive recombinant bacteria in the step (3), suspending the positive recombinant bacteria by using 10ml of Tris-HCl buffer solution (pH8.0), carrying out ultrasonic disruption for 5min under the condition of 39W, preparing and obtaining a cell-free extract (namely suspension after ultrasonic disruption), centrifuging, collecting supernate, and carrying out enzyme activity determination. The final concentration composition of the reaction system is as follows: Tris-HCl buffer (pH8.0), 0.1mM PLP, 20mM PPO, 60mM L-glutamic acid, in a 5mL system. Reaction conditions are as follows: reacting at 37 deg.C and 600r/min, boiling in ice bath for 5min to terminate the reaction, centrifuging at 12000r/min for 2min, and collecting 200 μ L supernatant for derivatization; the L-PPT concentration was determined by HPLC. The analytical column was a C18 column (250X 4.6mm, 5 μm) (Elite analytical instruments, Inc., Dalian, China). An LC-U3000 liquid chromatograph equipped with a fluorescence detector was used. Definition of enzyme activity: the amount of enzyme required to produce 1. mu. mol L-PPT per minute at 37 ℃ and pH8.0 is defined as one enzyme activity unit (U).

The results of this example are: high-throughput screening is applied, 181 recombinant transformation bacteria are screened for the first time, 4 mutant strains with improved enzyme activity are screened out, and then enzyme activity is measured, and specific results are shown in table 1. Analysis confirms that the reason that the enzyme activity of the other 177 strains of recombinant bacteria is kept unchanged or reduced is that proline (P) at position 52 is mutated into W, L, V and other amino acids except F.

Table 1: enzyme activity determination of single-point mutation recombinant bacteria

The mutant CkTA-P52V with the most obvious enzyme activity improvement is recorded as CkTA1, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA1 is obtained.

Example 2: construction and screening of CkTA two-site mutant

A site-directed mutagenesis primer is designed according to the single mutant CkTA1 sequence constructed in the example 1, and a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA1 is taken as a template, and a single mutation is introduced into the 71 th site, wherein the primer is as follows:

forward primer GTTATTTGGTGCANNKCATCCGGTG (base mutation underlined)

Reverse primer GTAACACCGGATGNNKTGCACC (mutated bases underlined)

And (3) PCR reaction system: 2X Phanta Max Buffer (containing Mg)²⁺)25 μ L of dNTPs 10mM, forward primer 2 μ L, reverse primer 2 μ L, template DNA1 μ L, Phanta Max Super-Fidelity DNApolymerase 50U, ddH was added₂O to 50. mu.L.

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 54 ℃ for 6.5min)30 cycles; 5min at 72 ℃.

Coli BL21(DE3) competent cells were transformed with PCR products and mutants were primary screened using bromothymol blue high throughput color development (same as example 1).

The wet cells were subjected to ultrasonication, and the enzyme activity of the preliminarily screened positive mutant was measured (same as in example 1).

The results of this example are: the 186 recombinant transformed bacteria are screened primarily by a high-throughput screening method, 5 mutant strains with improved enzyme activity are screened out, and then the enzyme activity is measured, and specific results are shown in table 2. Analysis proves that the reason that the enzyme activity of the rest 181 recombinant strains is kept unchanged or reduced is that the 71 th aspartic acid (D) is mutated into W, L, V, F and other amino acids except S.

Table 2: enzyme activity determination of double-point mutation recombinant bacteria

The mutant CkTA1-D71L with the most improved enzyme activity is recorded as CkTA2, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA2 is obtained.

Example 3: construction and screening of CkTA three-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA2 sequence constructed in the example 2, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA2 is taken as a template, a single mutation is introduced to the 81 th site, and the primer is:

forward primer CAACGGGTGNNKACCATCC (mutated bases underlined)

Reverse primer GTTTGGATGGTNNKCACCCG (mutated bases underlined)

And (3) PCR reaction system: 2X Phanta Max Buffer (containing Mg)²⁺)25 μ L of dNTPs 10mM, forward primer 2 μ L, reverse primer 2 μ L, template DNA1 μ L, Phanta Max Super-Fidelity DNA Polymerase 50U, ddH was added₂O to 50. mu.L.

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 60 ℃ for 6.5min)30 cycles; 5min at 72 ℃.

The positive mutants obtained by high-throughput primary screening were subjected to ultrasonication and enzyme activity assay (same as example 1).

The results of this example are: and (3) primarily screening 175 recombinant transformation bacteria, screening 2 mutant strains with improved enzyme activity, and then measuring the enzyme activity of the mutant strains, wherein specific results are shown in a table 3. Analysis confirms that the reason that the enzyme activity of the other 173 strains of recombinant bacteria is kept unchanged or reduced is that the 81 th alanine (A) is mutated into other amino acids except W and S.

Table 3: enzyme activity determination of three-point mutation recombinant bacteria

The mutant CkTA2-A81S with the most improved enzyme activity is recorded as CkTA3, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA3 is obtained.

Example 4: construction and screening of CkTA four-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA3 sequence constructed in the example 3, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA3 is taken as a template, a single mutation is introduced to the 153 th site, and the primer is:

forward primer CAACCCTGAATNNKCTGCCGG (base mutation underlined)

Reverse primer GGGCCGGCAGNNKATTCAG (base mutation underlined)

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 56 ℃ for 6.5min) for 30 cycles; 5min at 72 ℃.

The results of this example are: primary screening is carried out on 187 recombinant transformation bacteria, 7 mutant strains with improved enzyme activity are screened out, and then enzyme activity is measured, and specific results are shown in table 4. Analysis confirms that the reason that the enzyme activity of the other 180 strains of recombinant bacteria is kept unchanged or reduced is that the 153 th threonine (T) is mutated into W, V, F, S, R, K and other amino acids except I.

Table 4: enzyme activity determination of four-point mutation recombinant bacteria

The mutant CkTA3-T153R with the most improved enzyme activity is recorded as CkTA4, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA4 is obtained.

Example 5: construction and screening of CkTA five-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA4 sequence constructed in the example 4, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA4 is taken as a template, a single mutation is introduced into the 168 th site, and the primer is:

forward primer CCGTGTTGTCATNNKCCGACCGGC (the mutated base is underlined) Reverse primer CTGCGCCGGTCGGNNKATGACAAC (base mutation underlined)

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 62 ℃ for 6.5min)30 cycles; 5min at 72 ℃.

The results of this example are: and (3) primarily screening 193 recombinant transformation bacteria, screening 4 mutant strains with improved enzyme activity, and then measuring the enzyme activity of the mutant strains, wherein specific results are shown in a table 5. Analysis proves that the reason that the enzyme activity of the 189 strains of recombinant bacteria is kept unchanged or reduced is that the 168 th asparagine (N) is mutated into F, S, P and other amino acids except K.

Table 5: enzyme activity determination of five-point mutation recombinant bacteria

The mutant CkTA4-N168P with the most improved enzyme activity is recorded as CkTA5, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA5 is obtained.

Example 6: construction and screening of CkTA six-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA5 sequence constructed in the example 5, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA5 is taken as a template, a single mutation is introduced into the 324 th site, and the primer is:

forward primer CAGGTCGTAATTTTNNKTATTTACTGC (base mutation underlined)

Reverse primer CTGTTGCAGTAAATANNKAAAATTACGAC (base mutation underlined)

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 55 ℃ for 6.5min) for 30 cycles; 5min at 72 ℃.

The primary screening positive mutants were sonicated and assayed for enzyme activity using sonication (same as in example 1).

The results of this example are: 276 recombinant transformation bacteria are screened out for the first time, 1 mutant strain with improved enzyme activity is screened out, and then the enzyme activity is measured, and the specific results are shown in table 6. The reason that the enzyme activity of the rest 275 recombinant strains is kept unchanged or reduced is determined by analyzing that the 324 th aspartic acid (D) is mutated into other amino acids out of V.

Table 6: enzyme activity determination of six-point mutation recombinant bacteria

The mutant CkTA5-D324V with the most improved enzyme activity is recorded as CkTA6, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA6 is obtained.

Example 7: construction and screening of CkTA seven-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA6 sequence constructed in the example 6, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA6 is taken as a template, a single mutation is introduced into the 341 th site, and the primer is:

forward primer GGACTGAGCGCANNKCAGGTTG (mutated bases underlined)

Reverse directionPrimer CGATCAACCTGNNKTGCGCTCAG (base mutation underlined)

PCR amplification conditions were 95 ℃ for 3 min; (95 ℃ for 15s, 50 ℃ for 15s, 52 ℃ for 6.5min) for 30 cycles; 5min at 72 ℃.

The results of this example are: 188 recombinant transforming bacteria are screened out for the first time, 5 mutant strains with improved enzyme activity are screened out, and then the enzyme activity is measured, and the specific results are shown in table 7. Analysis proves that the reason that the enzyme activity of the rest 183 recombinant strains is kept unchanged or reduced is that the 341 th alanine (A) is mutated into F, L, S, K and other amino acids except I.

Table 7: enzyme activity determination of seven-point mutation recombinant bacteria

The mutant CkTA6-A341L with the most improved enzyme activity is recorded as CkTA7, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA7 is obtained.

Example 8: construction and screening of CkTA eight-site mutant

A site-directed mutagenesis primer is designed according to the mutant CkTA7 sequence constructed in the example 7, a rapid PCR technology is utilized, a recombinant vector pET28b/CkTA7 is taken as a template, a single mutation is introduced to the 361 th site, and the primer is:

forward primer GCGGTCGTATGNNKGTTGCG (mutated bases underlined)

Reverse primer GACCCGCAACNNKCATACGAC (mutant base underlined)

PCR reaction system: 2X Phanta Max Buffer (containing Mg)²⁺)25 μ L of dNTPs 10mM, forward primer 2 μ L, reverse primer 2 μ L, template DNA1 μ L, Phanta Max Super-Fidelity DNApolymerase 50U, ddH was added₂O to 50. mu.L.

The results of this example are: and (3) primarily screening 180 recombinant transformation bacteria, screening 9 mutant strains with improved enzyme activity, and then measuring the enzyme activity of the mutant strains, wherein specific results are shown in a table 8. Analysis confirmed that the reason why the activity of the other 171 recombinant strains remained unchanged or decreased is that the 361 th cysteine (C) is mutated into W, L, V, F, S, R, P, K and other amino acids except I.

Table 8: enzyme activity determination of eight-point mutation recombinant bacteria

The mutant CkTA7-C361I with the most improved enzyme activity is recorded as CkTA8, and the recombinant strain E.coli BL21(DE3)/pET28b/CkTA8 is obtained.

Example 9: recombinant escherichia coli fermentation enzyme production

Recombinant bacteria E.coli BL21(DE3)/pET28b/CkTA1, E.coli BL 1(DE 1)/pET 28 1/CkTA 1, E.coli BL 1(DE 1)/pET 28/kTA 1/CkTA 1 are inoculated to a final concentration of 50. mu.g/mL liquid culture medium at LB 150 ℃ and LB 150 OD 37 ℃ and OD 37 ℃ respectively in examples 1-8₆₀₀About 0.6 to about 0.8, obtaining a seed solution; inoculating the seed liquid into a fresh LB liquid culture medium containing 50 mu g/mL kanamycin at a final concentration by 2% (v/v) inoculation amount at 37℃,150r/min culture OD₆₀₀And when the concentration is 0.6-0.8, adding IPTG with the final concentration of 1mM into the culture solution, performing induced expression for 12 hours at 28 ℃, centrifuging for 10 minutes at 4 ℃ and 8000r/min, discarding supernatant, washing wet thalli twice by using 0.85% physiological saline, and collecting the wet thalli for later use.

Example 10: determination of optimum temperature of catalytic enzyme

After wet cells of each recombinant strain prepared in example 9 were collected and sonicated for 5min under 39W, the disrupted mixture was centrifuged at 4 ℃ and 8000r/min for 10min to discard the precipitate, the supernatant was collected and purified using a nickel-NTA agarose gel column, the column was equilibrated with an equilibration buffer (20mM phosphate buffer, 300mM NaCl, 20mM imidazole, pH8.0), and then eluted with an eluent (50mM phosphate buffer, 300mM NaCl, 500mM imidazole, pH8.0), and the corresponding eluates were collected according to the signal response of an ultraviolet detector, and thus, the respective pure enzyme solutions were obtained.

The above-mentioned pure enzyme solution was used as an enzyme for conversion, and the optimum reaction temperature of the enzyme was measured. The specific operation is as follows: to 100mM Tris-HCl buffer (pH8.0), 20mM PPO, 60mM L-glutamic acid, 0.1mM PLP and 1mL of pure enzyme solution were added, and the total volume was 5 mL. At different conversion temperatures: TA activity was measured at 32-77 deg.C (same procedure as in example 1), and the results are shown in FIG. 1.

As can be seen from the figure, the optimum reaction temperature of E.coli BL21(DE3)/pET28b/CkTA8 is 67 ℃, which is 10 ℃ higher than that of the original enzyme CkTA, and belongs to the highest value reported in the literature.

Example 11: selection of amino donors

The mutant CkTA8 purified enzyme solution of example 10 was used as an enzyme for transformation, and the optimum amino group donor of the enzyme was optimized. The specific operation is as follows: to 100mM Tris-HCl buffer (pH8.0), 20mM PPO, 60mM L-amino acid, 0.1mM PLP and 1mL of pure enzyme solution were added, and the total volume was 5 mL. The L-amino acids include, specifically, L-glutamic acid, L-alanine, L-aspartic acid, L-phenylalanine, L-methionine, L-proline, L-lysine, L-histidine, L-asparagine, L-valine, L-arginine, L-glutamine, L-tyrosine and L-tryptophan. The activity of TA in different amino donors was determined and the results are shown in FIG. 2.

As can be seen, the amino group donors of e.coli BL21(DE3)/pET28b/CkTA8 are very broad and include L-glutamic acid, L-alanine, L-proline, L-lysine, L-asparagine and L-valine.

Example 12: recombinant bacteria whole cell catalysis glufosinate-ammonium precursor ketone PPO asymmetric synthesis L-PPT according to the method of example 9, recombinant bacteria E.coli BL21(DE3)/pET28b/CkTA1, E.coli BL 1(DE 1)/pET 28 1/CkTA 1, E.coli BL 1(DE 1)/pET 28 CkTA 1/CkTA 1, E.coli BL 1(DE 1)/PPT 1/CkTA 1 are obtained, and the biological catalyst is prepared by using the wet ammonium phosphine precursor ketone as a biological substrate. 100mL of catalyst was prepared from 200mM Tris-HCl buffer (pH8.0) as the reaction medium, 200-600mM PPO, 3-fold molar L-glutamic acid amino group donor, 0.2mM PP, and 50g/L of wet cells. The system reacts for 6 hours at 67 ℃ and 600r/min, the concentration of L-PPT is periodically detected by HPLC, and the conversion rate is calculated. As can be seen from table 9, e.coli bl21(DE3)/pET28b/CkTA8 showed a substrate conversion of up to 100% at 4h, which is higher than the conversion of the original enzyme and other mutant enzymes, and also the highest level reported in the literature.

Table 9: comparison of conversion rates of glutamic acid as amino donor at different substrate concentrations

Sequence listing

<110> Zhejiang industrial university

<120> alpha-transaminase mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 389

<212> PRT

<213> Citrobacter koseri

<400> 1

Met Glu Arg Phe Lys Glu Asp Ser Arg Ser Asp Lys Val Asn Leu Ser

1 5 10 15

Ile Gly Leu Tyr Tyr Asn Glu Glu Gly Ile Ile Pro Gln Leu Lys Ala

20 25 30

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

35 40 45

Leu Tyr Leu Pro Met Glu Gly Leu Asn Thr Tyr Arg His Thr Ile Ala

50 55 60

Pro Leu Leu Phe Gly Ala Asp His Pro Val Leu Gln Gln Gln Arg Val

65 70 75 80

Ala Thr Ile Gln Thr Leu Gly Gly Ser Gly Ala Leu Lys Val Gly Ala

85 90 95

Asp Phe Leu Lys Arg Tyr Phe Pro Glu Ser Ala Val Trp Val Ser Asp

100 105 110

Pro Thr Trp Glu Asn His Ile Ala Ile Phe Glu Gly Ala Gly Phe Glu

115 120 125

Val Ser Thr Tyr Pro Trp Tyr Asp Asn Ala Thr Asn Gly Val Arg Phe

130 135 140

Asn Asp Leu Leu Ala Thr Leu Asn Thr Leu Pro Ala Arg Ser Ile Val

145 150 155 160

Leu Leu His Pro Cys Cys His Asn Pro Thr Gly Ala Asp Leu Thr His

165 170 175

Ser Gln Trp Asp Ala Val Ile Glu Ile Leu Lys Ala Arg Glu Leu Ile

180 185 190

Pro Phe Leu Asp Ile Ala Tyr Gln Gly Phe Gly Ala Gly Met Glu Asp

195 200 205

Asp Ala Tyr Ala Ile Arg Ala Ile Ala Ser Ala Gly Leu Pro Ala Leu

210 215 220

Val Ser Asn Ser Phe Ser Lys Ile Phe Ser Leu Tyr Gly Glu Arg Val

225 230 235 240

Gly Gly Leu Ser Val Val Cys Glu Asp Ala Glu Ala Ala Gly Arg Val

245 250 255

Leu Gly Gln Leu Lys Ala Thr Val Arg Arg Asn Tyr Ser Ser Pro Pro

260 265 270

Asn Phe Gly Ala Gln Val Val Ala Ala Val Leu Asn Asp Glu Ala Leu

275 280 285

Lys Ala Ser Trp Leu Ala Glu Val Glu Ala Met Arg Thr Arg Ile Leu

290 295 300

Ala Met Arg Gln Glu Leu Val Asn Val Leu Asn Ala Glu Ile Pro Gly

305 310 315 320

Arg Asn Phe Asp Tyr Leu Leu Gln Gln Arg Gly Met Phe Ser Tyr Thr

325 330 335

Gly Leu Ser Ala Ala Gln Val Asp Arg Leu Arg Asp Glu Phe Gly Val

340 345 350

Tyr Leu Ile Ala Ser Gly Arg Met Cys Val Ala Gly Leu Asn Ser Gly

355 360 365

Asn Val Gln Arg Val Ala Lys Ala Phe Ala Ala Val Met Leu Glu His

370 375 380

His His His His His

385

<210> 3

<211> 1167

<212> DNA

<213> Citrobacter koseri

<400> 3

atggagcgtt ttaaagagga ttcgcgcagc gataaagtta atttaagcat tggtctttac 60

tacaacgagg agggtattat tccgcagctg aaagccgttg cagaagccga agcacgtatt 120

aatgcacagc cacatggtgc cagcctgtat ctgccgatgg aaggattaaa tacctatcgt 180

cacaccattg cacccctgtt atttggtgca gatcatccgg tgttacagca gcaacgggtg 240

gcaaccatcc aaacattagg aggtagcggt gccctgaaag ttggcgcaga ttttttaaaa 300

agatactttc ctgagagcgc agtttgggtt agtgatccga cctgggaaaa tcatattgca 360

atttttgaag gcgccggatt tgaagttagt acctatccgt ggtatgacaa tgcaacgaat 420

ggggttcgtt ttaatgatct gctggcaacc ctgaataccc tgccggcccg tagcattgtt 480

ctgctgcatc cgtgttgtca taatccgacc ggcgcagatc tgacccatag tcagtgggat 540

gcggttattg aaattttaaa agcaagagaa ctgatccctt ttctggatat tgcctatcaa 600

ggttttggtg cggggatgga agatgatgca tatgcaattc gtgctattgc gagcgcgggt 660

ctgccggcac tggtttcaaa tagctttagc aaaattttct ccctgtatgg tgaacgtgtt 720

ggggggctga gcgttgtgtg tgaagatgcc gaagcagcag gtcgggtttt aggtcagctg 780

aaggcaactg ttcgtcgtaa ttatagcagc ccgcctaatt ttggtgctca ggttgttgca 840

gcggttttaa atgatgaagc gctgaaggcg agttggctgg cagaagttga agcaatgcgg 900

acccgcattt tagcaatgcg gcaagaatta gttaatgttc tgaatgcaga aatcccaggt 960

cgtaattttg attatttact gcaacagcgg ggtatgttta gctataccgg actgagcgca 1020

gcacaggttg atcgtttacg tgatgaattt ggcgtttatc tgattgcaag cggtcgtatg 1080

tgtgttgcgg gtctgaatag cggtaatgtt cagcgtgttg ccaaagcatt tgcagcagtt 1140

atgctcgagc accaccacca ccaccac 1167

<210> 3

<211> 389

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Arg Phe Lys Glu Asp Ser Arg Ser Asp Lys Val Asn Leu Ser

1 5 10 15

Ile Gly Leu Tyr Tyr Asn Glu Glu Gly Ile Ile Pro Gln Leu Lys Ala

20 25 30

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

35 40 45

Leu Tyr Leu Val Met Glu Gly Leu Asn Thr Tyr Arg His Thr Ile Ala

50 55 60

Pro Leu Leu Phe Gly Ala Leu His Pro Val Leu Gln Gln Gln Arg Val

65 70 75 80

Ser Thr Ile Gln Thr Leu Gly Gly Ser Gly Ala Leu Lys Val Gly Ala

85 90 95

Asp Phe Leu Lys Arg Tyr Phe Pro Glu Ser Ala Val Trp Val Ser Asp

100 105 110

Pro Thr Trp Glu Asn His Ile Ala Ile Phe Glu Gly Ala Gly Phe Glu

115 120 125

Val Ser Thr Tyr Pro Trp Tyr Asp Asn Ala Thr Asn Gly Val Arg Phe

130 135 140

Asn Asp Leu Leu Ala Thr Leu Asn Arg Leu Pro Ala Arg Ser Ile Val

145 150 155 160

Leu Leu His Pro Cys Cys His Pro Pro Thr Gly Ala Asp Leu Thr His

165 170 175

Ser Gln Trp Asp Ala Val Ile Glu Ile Leu Lys Ala Arg Glu Leu Ile

180 185 190

Pro Phe Leu Asp Ile Ala Tyr Gln Gly Phe Gly Ala Gly Met Glu Asp

195 200 205

Asp Ala Tyr Ala Ile Arg Ala Ile Ala Ser Ala Gly Leu Pro Ala Leu

210 215 220

Val Ser Asn Ser Phe Ser Lys Ile Phe Ser Leu Tyr Gly Glu Arg Val

225 230 235 240

Gly Gly Leu Ser Val Val Cys Glu Asp Ala Glu Ala Ala Gly Arg Val

245 250 255

Leu Gly Gln Leu Lys Ala Thr Val Arg Arg Asn Tyr Ser Ser Pro Pro

260 265 270

Asn Phe Gly Ala Gln Val Val Ala Ala Val Leu Asn Asp Glu Ala Leu

275 280 285

Lys Ala Ser Trp Leu Ala Glu Val Glu Ala Met Arg Thr Arg Ile Leu

290 295 300

Ala Met Arg Gln Glu Leu Val Asn Val Leu Asn Ala Glu Ile Pro Gly

305 310 315 320

Arg Asn Phe Val Tyr Leu Leu Gln Gln Arg Gly Met Phe Ser Tyr Thr

325 330 335

Gly Leu Ser Ala Leu Gln Val Asp Arg Leu Arg Asp Glu Phe Gly Val

340 345 350

Tyr Leu Ile Ala Ser Gly Arg Met Ile Val Ala Gly Leu Asn Ser Gly

355 360 365

Asn Val Gln Arg Val Ala Lys Ala Phe Ala Ala Val Met Leu Glu His

370 375 380

His His His His His

385

<210> 4

<211> 1167

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggagcgtt ttaaagagga ttcgcgcagc gataaagtta atttaagcat tggtctttac 60

tacaacgagg agggtattat tccgcagctg aaagccgttg cagaagccga agcacgtatt 120

aatgcacagc cacatggtgc cagcctgtat ctggtgatgg aaggattaaa tacctatcgt 180

cacaccattg cacccctgtt atttggtgca ctgcatccgg tgttacagca gcaacgggtg 240

agtaccatcc aaacattagg aggtagcggt gccctgaaag ttggcgcaga ttttttaaaa 300

agatactttc ctgagagcgc agtttgggtt agtgatccga cctgggaaaa tcatattgca 360

atttttgaag gcgccggatt tgaagttagt acctatccgt ggtatgacaa tgcaacgaat 420

ggggttcgtt ttaatgatct gctggcaacc ctgaataggc tgccggcccg tagcattgtt 480

ctgctgcatc cgtgttgtca tccgccgacc ggcgcagatc tgacccatag tcagtgggat 540

gcggttattg aaattttaaa agcaagagaa ctgatccctt ttctggatat tgcctatcaa 600

ggttttggtg cggggatgga agatgatgca tatgcaattc gtgctattgc gagcgcgggt 660

ctgccggcac tggtttcaaa tagctttagc aaaattttct ccctgtatgg tgaacgtgtt 720

ggggggctga gcgttgtgtg tgaagatgcc gaagcagcag gtcgggtttt aggtcagctg 780

aaggcaactg ttcgtcgtaa ttatagcagc ccgcctaatt ttggtgctca ggttgttgca 840

gcggttttaa atgatgaagc gctgaaggcg agttggctgg cagaagttga agcaatgcgg 900

acccgcattt tagcaatgcg gcaagaatta gttaatgttc tgaatgcaga aatcccaggt 960

cgtaattttg tgtatttact gcaacagcgg ggtatgttta gctataccgg actgagcgca 1020

ctgcaggttg atcgtttacg tgatgaattt ggcgtttatc tgattgcaag cggtcgtatg 1080

attgttgcgg gtctgaatag cggtaatgtt cagcgtgttg ccaaagcatt tgcagcagtt 1140

atgctcgagc accaccacca ccaccac 1167

Claims

1. An alpha-transaminase mutant, characterized in that the mutant is one of:

(1) the sequence is shown as SEQ ID NO: 1, mutating proline at position 52 of the amino acid shown in the specification into valine;

(2) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, and the 71 th aspartic acid is mutated into leucine;

(3) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, and the 81 th alanine is mutated into serine;

(4) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, the 81 th alanine is mutated into serine, and the 153 th threonine is mutated into arginine;

(5) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, the 81 th alanine is mutated into serine, the 153 th threonine is mutated into arginine, and the 168 th asparagine is mutated into proline;

(6) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, the 81 th alanine is mutated into serine, the 153 th threonine is mutated into arginine, the 168 th asparagine is mutated into proline, and the 324 th aspartic acid is mutated into valine;

(7) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, the 81 th alanine is mutated into serine, the 153 th threonine is mutated into arginine, the 168 th asparagine is mutated into proline, the 324 th aspartic acid is mutated into valine, and the 341 th alanine is mutated into leucine;

(8) the sequence is shown as SEQ ID NO: 1, the 52 th proline of the amino acid is mutated into valine, the 71 th aspartic acid is mutated into leucine, the 81 th alanine is mutated into serine, the 153 th threonine is mutated into arginine, the 168 th asparagine is mutated into proline, the 324 th aspartic acid is mutated into valine, the 341 th alanine is mutated into leucine, and the 361 th cysteine is mutated into isoleucine.

2. The α -transaminase mutant of claim 1, characterized in that the amino acid sequence of the α -transaminase mutant is as set forth in SEQ ID NO: 3, respectively.

3. Use of an alpha-transaminase mutant as claimed in claim 1 to catalyse the asymmetric synthesis of L-glufosinate from the precursor ketone of glufosinate-ammonium.

4. The use according to claim 3, characterized in that the use is: taking wet thalli obtained by fermenting and culturing recombinant genetic engineering bacteria containing the alpha-transaminase mutant coding gene or supernatant obtained by carrying out ultrasonic crushing on the wet thalli as a catalyst, taking glufosinate-ammonium precursor ketone PPO as a substrate, taking pyridoxal phosphate as a coenzyme, taking natural amino acid as an amino donor, reacting in a Tris-HCl buffer solution with the pH value of 8.0 at the temperature of 32-77 ℃ at the speed of 400-600 r/min, and after the reaction is finished, separating and purifying reaction liquid to obtain the L-glufosinate-ammonium.

5. The use according to claim 4, wherein the gene sequence encoding the mutant α -transaminase is as set forth in SEQ ID No. 4.

6. The use according to claim 4, wherein the initial concentration of the substrate in the reaction system is 20 to 800mM, the amount of the wet cells is 10 to 100g/L, and the amount of the coenzyme is 0 to 1 mM.