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

Abstract

The invention discloses a transaminase mutant and application of the transaminase mutant in asymmetric synthesis of L-glufosinate-ammonium. The transaminase mutant is obtained by carrying out single-point saturated mutation or iterative saturated mutation on 13th, 17th, 19th or 22th amino acids of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the transaminase mutant. The invention provides a novel high-activity transaminase mutant derived from salmonella, and the whole-cell relative enzyme activity of different mutants reaches 250.1%-397.9% compared with that before mutation.

Description

(I) Technical field

[0001] The invention relates to a (S)-transaminase mutant and application thereof in the asymmetric synthesis of L-phosphinothricin ammonium by biocatalysis of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid (PPO). (II) Background technology

[0002] Phosphinothricin (PPT; chemical name: 2-amino-4-[hydroxy(methyl)phosphonyl]butyrate ammonium or 4-[hydroxy(methyl)phosphonyl]-DL-homoalanine, chemical formula: C5H 15 N2O4P) is a low-toxic, high-efficiency, broad-spectrum organophosphorus herbicide developed by Germany's Hoechst (now part of Bayer) in the 1980s. Glufosinate, glyphosate and paraquat are known as the world's three major herbicides. Glufosinate inhibits plant glutamine synthetase, causing plant metabolism disorders and failure to synthesize chlorophyll, leading to death.

[0003] Glufosinate has two optical isomers, namely L-Glufosinate and D-Glufosinate, but only L-type has herbicidal activity, and is easily decomposed in soil, less toxic to humans and animals, has a wide weeding spectrum, and is environmentally friendly. At present, the glufosinate sold on the market is generally a racemic mixture. If the glufosinate product can be used in the form of pure optical isomers of L-configuration, the glufosinate dosage can be halved, and the discharge of D-Glufosinate (ineffective body) is gradually eliminated, which is of great significance for improving atom economy, reducing use costs, and alleviating environmental pressure. Therefore, efficient synthesis of L-Glufosinate is an important task.

[0004] There are two main methods for preparing chiral pure L-glufosinate ammonium: chemical method and biological method. The chemical method includes chemical resolution method and chemical synthesis method. However, these processes require the use of chiral resolution reagents, multiple resolutions, expensive catalysts, complex reaction routes and other disadvantages, making it difficult to achieve large-scale application. In contrast, the biocatalytic method has the advantages of mild reaction conditions, high stereoselectivity, and high yield, and is an important trend in the industrial preparation of L-glufosinate ammonium.

[0005] Bioenzymatic catalysis mainly includes kinetic resolution and asymmetric synthesis. The theoretical yield of kinetic resolution does not exceed 50%, while the theoretical yield of asymmetric synthesis can reach 100%. The method of preparing L-phosphinothricin by biocatalysis includes amidase, protease, nitrilase, and transaminase, among which amidase is a chiral resolution method, and the D-configuration by-product must be recovered. At present, the activity of nitrilase and protease is not very high, while transaminase is the most efficient and economical method in the preparation of chiral amines. Its asymmetric synthesis of L-phosphinothricin does not require the provision of expensive cofactors such as NADPH, and has higher atom economy.

[0006] Transaminases (TA) are a class of PLP-dependent enzymes that can catalyze the transfer of amino groups between amino donors and amino acceptors. According to the location of the transferred amino group, transaminases can be roughly divided into α-transaminases and ω-transaminases, but the defect of the reaction is that its equilibrium constant is small, and an excess of amino donors or a by-product removal system is usually used to promote product generation. Patent CN1349561A reports an aspartate aminotransferase (Asp-TA) that uses aspartic acid as an amino donor to synthesize L-phosphinothricin ammonium, which is not only inefficient, but also requires 80°C under reaction conditions. International patent WO2017151573A1 uses L-glutamic acid as an amino donor, and there is still a large amount of residue after the reaction, and its by-product α-ketoglutaric acid is similar in structure to the product L-phosphinothricin ammonium, which makes it difficult to separate and purify the product.

[0007] L-alanine and isopropylamine are commonly used amino donors for transaminase, which are not only low in cost but also easy to separate and purify. Isopropylamine is the cheapest and has the smallest molecular weight, but only a few transaminases can utilize it. Therefore, L-alanine is the only choice for most transamination reactions, and the removal method of its byproduct pyruvate is mature, which has great industrial application value.

[0008] Patent CN105603015B completely converts 100mM PPO into L-phosphinothricin by adding excessive L-alanine, resulting in 300mM L-alanine remaining in the system at the end, which not only increases the donor cost, but also increases the difficulty of product separation and purification, which is not conducive to production. Patent CN 113621592 A obtains a mutant ABAT2-Y138F by modifying the transaminase active center. It catalyzes 20mM substrate PPO, requires 70mM L-alanine, and adds 20g / L wet bacteria to react at 40°C for 10h. The conversion rate reaches 99%, the substrate concentration is low, the donor dosage is large, and the reaction time is long.

[0009] Therefore, it is very important to develop a highly active mutant strain that uses L-alanine as an amino donor to asymmetric synthesize L-phosphinothricin. (III) Summary of the invention

[0010] The present invention aims to provide a transaminase mutant and its application in the asymmetric synthesis of L-phosphinothricin ammonium, mutate the distal site of the transaminase, construct different mutants to improve the catalytic activity of the transaminase SeTA to L-alanine, so as to improve the catalytic efficiency and reduce the industrial production cost.

[0011] The technical solution adopted by the present invention is:

[0012] The present invention provides a transaminase mutant derived from Salmonella enterica (WP_001095559.1), wherein the transaminase mutant is obtained by subjecting the 13th, 17th, 19th or 22nd amino acid of the amino acid sequence shown in SEQ ID NO.1 to single-point saturation mutation or iterative saturation mutation. The nucleotide sequence of the coding gene corresponding to the amino acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.2.

[0013] Preferably, the amino acid sequence of the amino acid transaminase mutant shown in SEQ ID NO.1 is mutated to one of the following: asparagine at position 13 is substituted with methionine (N13M), the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; (2) arginine at position 17 is substituted with valine (R17V), the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; (3) valine at position 19 is substituted with leucine (V19L), the amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8; (4) isoleucine at position 22 is substituted with aspartic acid (I22D), the amino acid sequence is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10; (5) isoleucine at position 22 is substituted with aspartic acid and asparagine at position 13 is substituted with methionine (N13M / I22D), the amino acid sequence is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12; (6) the isoleucine at position 22 is substituted by aspartic acid, the asparagine at position 13 is substituted by methionine, and the valine at position 19 is substituted by leucine (N13M / V19L / I22D), the amino acid sequence is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14; (7) the isoleucine at position 22 is substituted by aspartic acid, the asparagine at position 13 is substituted by methionine, the valine at position 19 is substituted by leucine, and the arginine at position 17 is substituted by valine ((N13M / R17V / V19L / / I22D), the amino acid sequence is shown in SEQ ID NO.15, and the nucleotide sequence is shown in SEQ ID NO.16.

[0014] Due to the particularity of the amino acid sequence, any fragment or variant of the polypeptide of the amino acid sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, such as its conservative variant, biologically active fragment or derivative, as long as the homology of the polypeptide fragment or polypeptide variant with the aforementioned amino acid sequence is more than 95%, all belong to the scope of protection of the present invention. The changes may include the deletion, insertion or substitution of amino acids in the amino acid sequence; for conservative changes of the variant, the replaced amino acid has a similar structure or chemical property to the original amino acid, such as replacing isoleucine with leucine, and the variant may also have non-conservative changes, such as replacing glycine with tryptophan.

[0015] The present invention also relates to the coding gene of the transaminase mutant. The last three codons tga of the base sequences of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO.16 of the present invention are terminators and do not participate in the editing of amino acids.

[0016] Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, as long as it has more than 90% homology with the polynucleotide, belongs to the scope of protection of the present invention. The mutant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The mutant of this polynucleotide can be an allelic variant or an abiotic variant, including a substitution variant, a deletion variant and an insertion variant. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of encoding amino acids.

[0017] The present invention also relates to a recombinant vector containing the transaminase mutant encoding gene and a recombinant genetic engineering bacterium containing the recombinant vector. The recombinant vector is based on pET28b (+), the insertion site is between Nco I and Not I, and the host bacteria of the recombinant genetic engineering bacterium is preferably Escherichia coli BL21 (DE3).

[0018] The invention designs mutation primers for site-directed mutagenesis according to the transaminase (Se_WT) shown in SEQ ID NO.1, uses a cloning vector carrying the transaminase as a template to perform site-directed mutagenesis to construct a mutant, uses a plasmid pET28b (+) or a vector capable of expressing the enzyme as an expression vector, transforms the recombinant plasmid into E. coli BL21 (DE3) cells or a host cell capable of expressing the enzyme, and cultures positive single clones after high-throughput screening and verification to obtain wet cells of recombinant bacteria containing the mutant of the invention.

[0019] The present invention also provides an application of the aminotransferase mutant in the asymmetric synthesis of L-phosphinothricin ammonium from 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid (PPO) by biocatalysis, wherein the application comprises: using wet bacteria obtained by fermentation and culture of a recombinant genetic engineering bacterium containing the aminotransferase mutant encoding gene or pure enzyme solution obtained by ultrasonically crushing the wet bacteria and purifying the wet bacteria with a nickel column as a catalyst, using 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid as a substrate, using pyridoxal phosphate (PLP) as a coenzyme, and using the natural amino acid L-alanine as an amino donor, forming a conversion system in a buffer solution of pH 7.0 to 9.0 (preferably pH 8.5), reacting at 30 to 50° C. (preferably 35° C.) and 600 to 800 rpm (preferably 600 rpm), and extracting the reaction solution to obtain L-phosphinothricin ammonium after the reaction is completed.

[0020] Furthermore, in the transformation system, the initial concentration of the substrate added is 20-80 mM, preferably 50 mM; the amount of wet bacteria is 5-20 g / L, preferably 10 g / L; the amount of coenzyme is 0.05-0.5 mM, preferably 0.1 mM; the amount of L-alanine is 50 mM to 150 mM, preferably 75 mM.

[0021] The wet bacterial cells obtained by fermentation culture of the engineered bacteria containing the transaminase mutant gene of the present invention can be prepared as follows: construct a recombinant vector containing the transaminase mutant gene with excellent catalytic activity, transform the recombinant vector into E. coli BL21 (DE3), induce expression of the obtained recombinant genetic engineered bacteria, and separate the wet bacterial cells from the culture fluid. Specifically, the engineered bacteria containing the transaminase mutant gene are inoculated into an LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C and 200rpm for 10-12h, and then inoculated into a fresh TB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C and 180rpm until the bacterial OD reaches 0. 600When the pH value reaches 0.4-0.6, add IPTG with a final concentration of 0.1 mM, induce and culture at 28°C for 12 hours, centrifuge at 4°C and 8000 rpm for 10 minutes, discard the supernatant, and collect the wet bacteria. The TB culture medium consists of 12 g / L tryptone, 24 g / L yeast powder, 12.54 g / L disodium hydrogen phosphate, 2.31 g / L sodium dihydrogen phosphate, 5 g / L glycerol, and the solvent is water.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) The enzyme activity of the transaminase mutant was significantly improved

[0024] The invention provides a novel high-activity transaminase mutant derived from Salmonella. Compared with that before mutation, the whole cell relative enzyme activity of the transaminase mutant reaches 250.1% to 397.9%.

[0025] (2) The space-time yield of L-phosphinothricin synthesized by the aminotransferase mutant was significantly improved

[0026] When reacting with 50mM substrate, the time for Se_WT to reach reaction equilibrium is 18h, and the time for recombinant transaminase mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D to reach reaction equilibrium is 3-6h; at equilibrium, the yield of L-phosphinothricin is 41.5%, and the product ee is ≥99.9%. Compared with the wild-type transaminase Se_WT, the reaction time of mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D is shortened by 12-15h, and the time-space yield is increased by 3-5 times. These mutants create good application prospects for the asymmetric synthesis of L-phosphinothricin by transaminase using L-alanine as an amino donor. (IV) Description of the drawings

[0027] Figure 1 This is the gel electrophoresis diagram of the recombinant transaminase Se_WT and its mutant PCR products in Example 1; a represents a single mutant, and b represents a combined mutant.

[0028] Figure 2 This is a bar graph showing the relative activities of the transaminase Se_WT and its single-point saturation mutants in Example 1.

[0029] Figure 3 This is a bar graph showing the relative activities of the transaminase Se_WT and its iterative saturated mutants in Example 1.

[0030] Figure 4The SDS-PAGE electrophoresis diagram of the crude transaminase enzyme solution Se_WT and its mutants in Example 3; a represents a single mutant, and b represents a combined mutant.

[0031] Figure 5 This is a reaction process diagram of the synthesis of L-phosphinothricin by transaminase Se_WT and its mutants in Example 5. (V) Specific implementation methods

[0032] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] The LB liquid culture medium used in the present invention comprises: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L NaCl. The LB plate comprises 20 g / L agar added to the LB liquid culture medium.

[0034] TB liquid culture medium composition: tryptone 12g / L, yeast powder 24g / L, disodium hydrogen phosphate 12.54g / L, sodium dihydrogen phosphate 2.31g / L, glycerol 5g / L.

[0035] Example 1. Construction of transaminase mutants and recombinant Escherichia coli

[0036] 1. Single-site saturation mutation

[0037] The plasmid pET28b-Se_WT (CN 114921432 A) containing the recombinant transaminase from Salmonella enterica (WP_001095559.1) was extracted as a starting template, and single-point saturation mutations were performed on the asparagine at position 13, the arginine at position 17, the valine at position 19, and the isoleucine at position 22 of the wild-type Se_WT (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2). The whole plasmid was amplified by overlapping extension PCR, and the primer sequences for each site saturation mutation are shown in Table 1, N represents A / C / G / T, and K represents G / T.

[0038] SEQ ID NO.1

[0039] MNTNNALMQRRHNAVPRGVGQIHPIFAERAENCRVWDVEGREYLDFAGGIAVLNTGHLHPGIVSAVEAQLKKLSHTCFQVLAYEPYLALCERMNQKVPGDFAKKTLLVT TGSEAVENAVKIARAATKRSGAIAFSGAYHGRTHYTLSLTGKVHPYSAGMGLMPGHVYRALYPCPLHNISDDDDAIASIERIFKNDAAPEDIAAIIIEPVQGEGGFYAAS PAFMQRLRALCDQHGIMLIADEVQSGAGRTGTLFAMEQMGVAADITTTFAKSIAGGFPLAGVTGRADVMDAIAPGGLGGTYAGNPIACAAALAVLDIFEQENLLQKANTLGNTLRDGLMEIAETHREIGDVRGLGAMIAIELFENGDPGKPNAALTADIVTRAREKGLILLSCGPYYNILRILVPLTIEASQIRQGLEIIAQCFDEAKQALEHHHHHH.

[0040] The PCR amplification system was (total volume 25 μL): template DNA 0.5 μL, 2×Phanta Max Buffer 12.5 μL, dNTPs (10 mM each of dATP, dCTP, dGTP and dTTP) 1 μL, upstream and downstream mutant primers 0.5 μL each, Phanta Max Super-Fidelity DNA Polymerase 0.5 μL, and ddH2O 9.5 μL.

[0041] PCR reaction conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 s; (3) annealing at 60°C for 30 s; (4) extension at 72°C for 4 min, steps (2)-(4) for a total of 30 cycles, (5) complete extension at 72°C for 10 min, and (6) storage at 16°C.

[0042] 5 μL of each PCR product was subjected to 1% agarose gel electrophoresis analysis. The target band size was 6154 bp. The electrophoretic patterns of Se_WT, mutants Se_N13M, Se_R17V, Se_V19L, and Se_I22D were shown in Figure 1 As shown in a.

[0043] After verifying that the size of the amplified band is correct, add 1 μL of DpnI enzyme to the remaining PCR reaction solution, digest at 37°C for 2 hours to remove the template plasmid DNA, and inactivate at 65°C for 10 minutes. Then take 4-6 μL of the PCR product after template elimination and transform it into 100 μL E.coli BL21 (DE3) competent cells through chemical heat shock method, add LB liquid culture medium to recover for 1 hour, centrifuge and discard part of the supernatant, then blow and mix the cells and spread them on an LB plate containing 50 μg / mL kanamycin, and culture at 37°C overnight to obtain transformants. Pick 3 transformants from each plate and add them to LB liquid culture medium containing 50 μg / mL kanamycin. After culturing at 37°C for 12 hours, take the bacterial solution for sequencing, and use the method in Example 4 to test the enzyme activity of the transformants. The results are shown in Figure 2 As shown, the superior mutants Se_N13M (amino acid sequence such as SEQ ID NO.3, nucleotide sequence such as SEQ ID NO.4), Se_R17V (amino acid sequence such as SEQ ID NO.5, nucleotide sequence such as SEQ ID NO.6), Se_V19L (amino acid sequence such as SEQ ID NO.7, nucleotide sequence such as SEQ ID NO.8), and Se_I22D (amino acid sequence such as SEQ ID NO.9, nucleotide sequence such as SEQ ID NO.10) were screened out, and the relative activities of catalyzing the synthesis of L-phosphinothricin were 277.8%, 250.1%, 300.7%, and 369.3% of that of the wild type, respectively, and the ee≥99.9%.

[0044] Table 1. Single-site saturation mutagenesis primer sequences (5' to 3')

[0045]

[0046]

[0047] 2. Iterative saturation mutation

[0048] On the basis of the single-site saturation mutation, the single mutants were subjected to iterative saturation mutation, the primers are shown in Table 2, and the enzyme activity of the transformants was tested by the method of Example 4. The results are shown in Figure 3 As shown, the superior mutants Se_N13M / I22D (amino acid sequence such as SEQ ID NO.11, nucleotide sequence such as SEQ ID NO.12), Se_N13M / V19L / I22D (amino acid sequence such as SEQ ID NO.13, nucleotide sequence such as SEQ ID NO.14), and Se_N13M / R17V / V19L / I22D (amino acid sequence such as SEQ ID NO.15, nucleotide sequence such as SEQ ID NO.16) were screened and the gel electrophoresis of the corresponding saturation mutation PCR was shown in FIG. Figure 1In b, the relative activities of catalyzing the synthesis of L-phosphinothricin were 359.2%, 397.9% and 389.1% of the wild type, respectively, and the ee was ≥ 99.9%.

[0049] Table 2. Sequences of primers for iterative saturation mutagenesis (5' to 3')

[0050] Primer name Primer sequence (5' to 3') N13M / I22D-F TGCAGCGTCGTCACNNKGCTGTTCCGCGTG N13M / I22D-R GTGACGACGCTGCATCAGAGCGTTGTTG N13M / V19L / I22D-F GCTGTTCCGCGTGGTNNKGGTCAGGACCACCCGATC N13M / V19L / I22D-R ACCACGCGGAACAGCCATGTGACGACGCTGCA N13M / R17V / V19L / I22D-F CATGGCTGTTCCGNNKGGTCTAGGTCAG N13M / R17V / V19L / I22D-R CGGAACAGCCATGTGACGACGCTGCATC

[0051] The recombinant Escherichia coli used to screen and obtain transaminase in this example are E. coli BL21 (DE3) / pET28b-Se_N13M, E. coli BL21 (DE3) / pET28b-Se_R17V, E. coli BL21 (DE3) / pET28b-Se_V19L, E. coli BL21 (DE3) / pET28b-Se_I22D, E. coli BL21 (DE3) / pET28b-Se_N13M / I22D, E. coli BL21 (DE3) / pET28b-Se_N13M / V19L / I22D, and E. coli BL21 (DE3) / pET28b-Se_N13M / R17V / V19L / I22D.

[0052] Example 2: Induced expression and collection of recombinant Escherichia coli containing transaminase

[0053] The recombinant E. coli BL21 (DE3) / pET28b-Se_WT and the mutant obtained in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance, respectively, and cultured at 37 ° C, 200 rpm for 10-12 h, and then inoculated into fresh TB liquid medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37 ° C, 180 rpm until the bacterial OD 600 When the p-value reaches 0.4-0.6, add IPTG with an initial concentration of 0.1 M, induce culture at 28°C for 12 h, centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the recombinant Escherichia coli wet cells containing the target gene, which are stored at -20°C for later use.

[0054] Example 3: SDS-PAGE electrophoresis detection of transaminase

[0055] 1. SDS-PAGE electrophoresis of crude enzyme solution of single point mutant

[0056] The wet cells of E.coli BL21(DE3) / pET28b-Se_WT and the dominant single mutant cells E.coli BL21(DE3) / pET28b-Se_N13M, E.coli BL21(DE3) / pET28b-Se_R17V, E.coliBL21(DE3) / pET28b-Se_V19L, and E.coli BL21(DE3) / pET28b-Se_I22D collected by the method of Example 2 were resuspended in 20mM phosphate buffer (pH 8.0) and the cell concentration was adjusted to 10g / L. Ultrasonic disruption (40W, continuous disruption for 1.5min) was performed in a low-temperature ice bath, and the disrupted cell fluid was centrifuged at 12000rpm for 1min, and the supernatant was the crude transaminase enzyme solution. The SDS-PAGE electrophoresis of the crude enzyme solution is shown in FIG. Figure 4 As shown in a, transaminase Se_WT and its mutants have a total of 435 amino acids (with 6×His-tag), and their theoretical molecular weight is 46.7 kDa, which proves that the recombinant E. coli in Example 1 was successfully constructed.

[0057] 2. SDS-PAGE electrophoresis of pure enzyme solution of iterative combination mutants

[0058] The wet cells of Example 1 E.coli BL21 (DE3) / pET28b-Se_WT and the dominant iterative mutant cells E.coli BL21 (DE3) / pET28b-Se_N13M / I22D, E.coli BL21 (DE3) / pET28b-Se_N13M / V19L / I22D, and E.coli BL21 (DE3) / pET28b-Se_N13M / R17V / V19L / I22D collected by the method of Example 2 were washed once with a physiological saline (NaCl) with a mass fraction of 0.85%, centrifuged at 4°C and 8000rpm for 10min, and the cells were collected again. The collected cells were resuspended with 20mM phosphate buffer (pH8.0) and the cell concentration was adjusted to 10g / L. Ultrasonic disruption was performed in a low-temperature ice bath (40W, continuous 2s, 4s interval, continuous disruption for 40min), and the disrupted cell fluid was centrifuged at 12000rpm for 10min. The supernatant was the crude enzyme solution.

[0059] First, rinse the protein chromatography system and Ni-NTA affinity column (40×12.6mm, Bio-Rad, USA) with ultrapure water at a flow rate of 3mL / min for 15min; then rinse the Ni-NTA column with buffer A at a flow rate of 3mL / min until the protein analyzer mark is stable; after loading the crude enzyme solution at a flow rate of 3mL / min, elute with buffer C at a flow rate of 2mL / min for 5 column volumes; then elute with buffer B at a rate of 2mL / min for 5 column volumes and collect the eluate. Subsequently, the collected eluate is placed in a dialysis bag of 8000-14000Da in size, and dialyzed in an ice bath in a pH 8.0, 20mM phosphate buffer for 12h for desalting. The buffer is changed every 6h, and the retentate is the pure enzyme solution of the corresponding transaminase; after appropriate dilution, SDS-PAGE electrophoresis is performed, such as Figure 4 As shown in b, the aminotransferase Se_WT and its mutants have a total of 435 amino acids (with 6×His-tag), and their theoretical molecular weight is 46.7 kDa, which proves that the recombinant E. coli in Example 1 was successfully constructed.

[0060] Buffer A: 20 mM Na2HPO4-NaH2PO4, 300 mM NaCl, pH 8.0.

[0061] Buffer B: 20 ​​mM Na2HPO4-NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0;

[0062] Buffer C: 20 mM Na2HPO4-NaH2PO4, 300 mM NaC1, 100 mM imidazole, pH 8.0.

[0063] Example 4: Determination of relative enzyme activity of whole cells

[0064] The wet bacteria prepared by the method of Example 2 was used as a catalyst, PPO was used as an acceptor substrate, and L-alanine was used as a donor for the transamination reaction.

[0065] Definition of transaminase activity unit (U): The amount of bacteria required to catalyze the substrate PPO to produce 1 μmoL product L-PPT within 1 min at 35°C and pH 8.5 is defined as 1U.

[0066] Relative enzyme activity: Se_WT enzyme activity was taken as 100% to calculate the relative enzyme activities of other mutant enzymes.

[0067] Reaction system (1mL): 50mM PPO, 75mM L-alanine, 0.1mM PLP, 10g / L (WCW) bacteria, reaction medium is Tris-HCl buffer (50mM, pH 8.5). The catalytic reaction is carried out at 35°C, 600rpm, 200μL is sampled regularly, 5μL 6M HCl is added to terminate the reaction, centrifuged at 12000rpm for 1min, the supernatant is taken for appropriate dilution, and the peak area of ​​L-glufosinate is detected by high performance liquid chromatography after derivatization. The L-glufosinate content of the product is calculated according to the standard curve of the peak area and concentration of the L-glufosinate standard under the same detection conditions, and then the enzyme activity is calculated.

[0068] Product L-phosphinothricin ammonium detection: Thermo Fisher U3000 liquid chromatograph equipped with a fluorescence detector, chromatographic column C18 column (4.6×250 mm, Acchrom, China); mobile phase was methanol: 50 mM ammonium acetate buffer (pH 5.7), volume ratio was 10:90; flow rate was 1.0 mL / min; detection wavelength Ex=350 nm, Em=460 nm; injection volume was 10 μL; column temperature was 35°C. The retention times of L-phosphinothricin and D-phosphinothricin were 10.6 minutes and 12.6 minutes, respectively.

[0069] Example 5. Application of mutants in L-PPT production (L-PPT yield)

[0070] The wet bacteria containing transaminase Se_WT, Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D prepared by the method in Example 2 were used as catalysts, PPO was used as the acceptor substrate, and L-alanine was used as the donor to determine the synthesis process of L-phosphinothricin.

[0071] Reaction system (10mL): 50mM PPO, 75mM L-alanine, 0.1mM PLP, 10g / L (WCW) bacteria, reaction medium is Tris-HCl buffer (50mM, pH 8.5). The catalytic reaction was carried out at 35°C and 600rpm. 200μL of samples were taken at 0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 18h, and 24h, and 5μL of 6M HCl was added to terminate the reaction. The supernatant was taken for appropriate dilution and detected by the method of Example 4 after derivatization. The results are shown in FIG. Figure 5 .

[0072] The process of the synthesis of L-phosphinothricin catalyzed by transaminase Se_WT and its mutants showed that the time for Se_WT to reach reaction equilibrium was 18h, the yield of L-phosphinothricin was 41.5%, and the product ee ≥ 99.9%.

[0073] The time for Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D to reach reaction equilibrium was 3-6h, respectively; at equilibrium, the yield of L-phosphinothricin was 41.5%, and the product ee was ≥99.9%. Compared with the wild-type transaminase Se_WT, the reaction time of mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D was shortened by 12-15h, and the time-space yield was increased by 3-5 times. These mutants have created good application prospects for the asymmetric synthesis of L-phosphinothricin by transaminase using L-alanine as an amino donor.

Claims

1. A transaminase mutant, characterized in that: The transaminase mutant is obtained by subjecting the 13th, 17th, 19th or 22nd amino acid of the amino acid sequence shown in SEQ ID NO.1 to single-point saturation mutation or iterative saturation mutation.

2. The aminotransferase mutant according to claim 1, characterized in that The amino acid transaminase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated to one of the following: (1) asparagine at position 13 is substituted by methionine; (2) arginine at position 17 is substituted by valine; (3) valine at position 19 is substituted by leucine; (4) isoleucine at position 22 is substituted by aspartic acid; (5) isoleucine at position 22 is substituted by aspartic acid and asparagine at position 13 is substituted by methionine; (6) isoleucine at position 22 is substituted by aspartic acid, asparagine at position 13 is substituted by methionine, and valine at position 19 is substituted by leucine; (7) isoleucine at position 22 is substituted by aspartic acid, asparagine at position 13 is substituted by methionine, valine at position 19 is substituted by leucine, and arginine at position 17 is substituted by valine.

3. A recombinant genetically engineered bacterium containing the gene encoding the aminotransferase mutant according to claim 1.

4. Use of the aminotransferase mutant according to claim 1 in the biocatalytic asymmetric synthesis of L-phosphinothricin from 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid.

5. The use according to claim 4, characterized in that The application is: using wet bacteria obtained by fermentation culture of recombinant genetic engineering bacteria containing the transaminase mutant encoding gene or pure enzyme liquid obtained by ultrasonic crushing of wet bacteria and purification by nickel column as a catalyst, using 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid as a substrate, using pyridoxal phosphate as a coenzyme, and using natural amino acid L-alanine as an amino donor, a conversion system is formed in a pH 7.0-9.0 buffer, reacting at 30-50°C and 600-800rpm, and after the reaction is completed, extracting the reaction liquid to obtain L-phosphinothricin.

6. The use according to claim 5, characterized in that In the transformation system, the initial concentration of the substrate added is 20-80 mM; the amount of wet bacteria is 5-20 g / L; the amount of coenzyme is 0.05-0.5 mM; and the amount of L-alanine is 50 mM to 150 mM.

7. The use according to claim 5, characterized in that The wet bacterial cells were prepared as follows: the engineered bacteria containing the transaminase mutant gene were inoculated into a LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 200 rpm for 10-12 hours, and then inoculated into a fresh TB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1% inoculum, and cultured at 37°C and 180 rpm until the bacterial OD 600 When the pH value reaches 0.4 to 0.6, IPTG with a final concentration of 0.1 mM is added, and the culture is induced at 28°C for 12 hours, and then centrifuged at 4°C and 8000 rpm for 10 minutes, the supernatant is discarded, and the wet bacteria are collected; the TB culture medium comprises: 12 g / L tryptone, 24 g / L yeast powder, 12.54 g / L disodium hydrogen phosphate, 2.31 g / L sodium dihydrogen phosphate, 5 g / L glycerol, and the solvent is water.

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