Transaminase mutants and their application in asymmetric synthesis of L-phosphinothricin

Abstract

The present invention discloses a transaminase mutant and its use in the asymmetric synthesis of L-phosphinothricin. The transaminase mutant is obtained by subjecting amino acids 13, 17, 19, or 22 of the amino acid sequence shown in SEQ ID NO. 1 to single-point saturation mutagenesis or iterative saturation mutagenesis. The present invention also provides a novel high-activity transaminase mutant derived from Salmonella. Compared to the premutation state, the whole-cell relative enzyme activity of different mutants reaches 250.1% to 397.9%.

Description

(1) Technical field

[0001] The present invention relates to a (S)-transaminase mutant and application thereof in the biocatalytic asymmetric synthesis of L-glufosinate from 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid (PPO). (2) Background technology

[0002] Phosphinothricin (PPT; chemical name 2-amino-4-[hydroxy(methyl)phosphonyl]butyrate or 4-[hydroxy(methyl)phosphonyl]-DL-homoalanine, chemical formula C5H 15 Developed in the 1980s by Hoechst (now part of Bayer), Glufosinate (N2O4P) is a low-toxic, highly effective, broad-spectrum organophosphorus herbicide. Glufosinate, along with glyphosate and paraquat, is considered one of the world's three major herbicides. Glufosinate inhibits plant glutamine synthetase, disrupting plant metabolism and preventing chlorophyll synthesis, leading to plant death.

[0003] Glufosinate-ammonium has two kinds of optical isomers, is respectively L-glutophosphine-ammonium and D-glutophosphine-ammonium, but only has L-type to have weeding activity, and easily decomposes in soil, less toxic to humans and animals, weeding spectrum is wide, environmentally friendly.At present, the glufosinate-ammonium sold on the market is generally all racemic mixture.If the glufosinate-ammonium product can be used with the pure optical isomer form of L-configuration, the glufosinate-ammonium consumption can be halved, gradually eliminate the discharge of D-glutophosphine-ammonium (ineffective body), this is significant for improving atom economy, reducing use cost, alleviating environmental pressure.Therefore efficient synthesis of L-glutophosphine-ammonium is an important task.

[0004] There are two main methods for preparing chirally pure L-glufosinate: chemical and biological. Chemical methods include chemical resolution and chemical synthesis. However, these processes require the use of chiral resolution reagents, require multiple resolutions, have expensive catalysts, and involve complex reaction routes, making them difficult to scale up. In contrast, biocatalytic methods offer advantages such as mild reaction conditions, high stereoselectivity, and high yields, making them a key trend in the industrial production of L-glufosinate.

[0005] Bioenzymatic catalysis primarily involves kinetic resolution and asymmetric synthesis. The theoretical yield of kinetic resolution is less than 50%, while the theoretical yield of asymmetric synthesis can reach 100%. Biocatalytic methods for preparing L-glufosinate include amidase, protease, nitrilase, and transaminase. Amidase is a chiral resolution method that requires recovery of D-configuration byproducts. Currently, the activity of nitrilase and protease is not very high. Transaminase, on the other hand, is the most efficient and economical method for preparing chiral amines. Its asymmetric synthesis of L-glufosinate does not require expensive cofactors such as NADPH and offers higher atom economy.

[0006] Transaminases (TAs) are a class of PLP-dependent enzymes that catalyze the transfer of amino groups between amino donors and amino acceptors. Depending on the location of the transferred amino group, transaminases can be roughly divided into α-transaminases and ω-transaminases, but the drawback 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 formation. Patent CN1349561A reports an aspartate aminotransferase (Asp-TA) that uses aspartic acid as an amino donor to synthesize L-glufosinate. Not only is the efficiency low, but the reaction temperature needs to reach 80°C. International patent WO2017151573A1 uses L-glutamic acid as an amino donor, and there is still a large amount of residue after the reaction. In addition, its by-product α-ketoglutaric acid has a similar structure to the product L-glufosinate, making it difficult to separate and purify the product.

[0007] L-alanine and isopropylamine are commonly used amino group donors for transaminases, offering low cost and easy product separation and purification. Isopropylamine is the cheapest and has the smallest molecular weight, but only a few transaminases can utilize it. Therefore, L-alanine is the preferred choice for most transamination reactions, and its removal of the byproduct pyruvate is well-established, making it a promising candidate for industrial applications.

[0008] Patent CN105603015B completely converts 100mM PPO into L-glufosinate by adding an excess of L-alanine, resulting in 300mM L-alanine remaining in the system at the end. This not only increases the donor cost but also makes product separation and purification more difficult, hindering production. Patent CN 113621592 A, by modifying the transaminase active center, obtained a mutant ABAT2-Y138F. Its catalysis of 20mM substrate PPO requires 70mM L-alanine. Adding 20g / L wet cells to the reaction at 40°C for 10 hours achieves a conversion rate of 99%. This low substrate concentration, high donor dosage, and long reaction time are the main reasons for this.

[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. (3) 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. The distal sites of the transaminase are mutated to construct different mutants to improve the catalytic activity of the transaminase SeTA towards L-alanine, thereby improving the catalytic efficiency and reducing industrial production costs.

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

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

[0013] Preferably, the amino acid sequence of the transaminase mutant is mutated into 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) 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 (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) 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 ((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 specificity of amino acid sequences, any fragment or variant of a polypeptide having an amino acid sequence as set forth 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, or SEQ ID NO.15, such as a conservative variant, biologically active fragment, or derivative thereof, as long as the polypeptide fragment or polypeptide variant has at least 95% homology with the aforementioned amino acid sequence, falls within the scope of protection of the present invention. Such alterations may include deletions, insertions, or substitutions of amino acids within the amino acid sequence; for conservative alterations of variants, the replaced amino acid has similar structural or chemical properties to the original amino acid, such as replacing isoleucine with leucine. Variants may also have non-conservative alterations, 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 amino acid editing.

[0016] Due to the specificity of nucleotide sequences, any variant of the polynucleotides set forth 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, or SEQ ID NO.16, as long as they share more than 90% homology with the polynucleotides, falls within the scope of protection of the present invention. A polynucleotide mutant refers to a polynucleotide sequence having one or more nucleotide alterations. Such polynucleotide mutants can be allelic or non-allelic variants, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may have one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded amino acid.

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

[0018] The present invention designs mutation primers for site-directed mutagenesis based on the transaminase (Se_WT) shown in SEQ ID NO.1, performs site-directed mutagenesis using a cloning vector carrying the transaminase as a template to construct a mutant, uses 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 cultured the positive single clones verified by high-throughput screening to obtain wet cells of recombinant bacteria containing the mutant of the present invention.

[0019] The present invention also provides an application of the transaminase mutant in the biocatalytic asymmetric synthesis of L-glufosinate from 2-carbonyl-4-[hydroxy(methyl)phosphonyl]-butyric acid (PPO). The application comprises the following steps: using wet cells obtained by fermentation and culturing a recombinant genetically engineered bacterium containing a gene encoding the transaminase mutant, or a pure enzyme solution obtained by ultrasonically crushing the wet cells and purifying them 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 group donor; forming a conversion system in a buffer solution having a pH of 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 after completion of the reaction to obtain L-glufosinate.

[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; and 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 culture fluid to obtain wet bacterial cells. Specifically, the engineered bacteria containing the transaminase mutant gene are inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C and 200 rpm for 10-12 hours, and then inoculated into 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 180 rpm until the bacterial OD reaches 0. 600When the pH reaches 0.4-0.6, IPTG is added to a final concentration of 0.1 mM. After induction culture at 28°C for 12 hours, the culture is centrifuged at 8000 rpm at 4°C for 10 minutes, the supernatant is discarded, and the wet cells are collected. The TB culture medium consists of 12 g / L tryptone, 24 g / L yeast extract, 12.54 g / L disodium hydrogen phosphate, 2.31 g / L sodium dihydrogen phosphate, 5 g / L glycerol, and water as the solvent.

[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 present 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 transaminase mutant was significantly improved

[0026] When reacting with 50 mM substrate, Se_WT reached equilibrium in 18 hours, while the recombinant transaminase mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D reached equilibrium in 3-6 hours. At equilibrium, the yield of L-glufosinate was 41.5%, with product ee ≥99.9%. Compared to the wild-type transaminase Se_WT, the mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D shortened the reaction time by 12-15 hours and increased the space-time yield by 3-5 times. These mutants offer promising application prospects for the asymmetric synthesis of L-glufosinate using L-alanine as an amino donor. (IV) Description of the accompanying drawings

[0027] Figure 1 Figure 2 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 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 saturation 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 with reference to 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 was composed of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. The LB plate was composed of 20 g / L agar added to the LB liquid culture medium.

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

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

[0036] 1. Single-point saturation mutation

[0037] The plasmid pET28b-Se_WT (CN 114921432 A), containing the recombinant transaminase from Salmonella enterica (WP_001095559.1), was used as a starting template. Single-site saturation mutagenesis was performed on the wild-type Se_WT (amino acid sequence shown in SEQ ID NO. 1, nucleotide sequence shown in SEQ ID NO. 2) at asparagine 13, arginine 17, valine 19, and isoleucine 22. Overlap extension PCR was used for full plasmid amplification. The primer sequences for each site saturation mutagenesis are shown in Table 1, where 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 as follows (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 2. Figure 1 As shown in a.

[0043] After verifying that the size of the amplified band is correct, 1 μL of DpnI enzyme is added to the remaining PCR reaction solution, and the template plasmid DNA is removed by enzyme digestion at 37°C for 2 hours, and then inactivated at 65°C for 10 minutes. Then 4-6 μL of the PCR product after template elimination is taken and transformed into 100 μL of E. coli BL21 (DE3) competent cells by chemical heat shock method, and LB liquid culture medium is added to recover for 1 hour. After centrifugation and discarding part of the supernatant, the cells are pipetted and mixed, and spread on an LB plate containing 50 μg / mL kanamycin, and cultured 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, the bacterial solution is taken for sequencing, and the enzyme activity of the transformants is tested using the method in Example 4. The results are shown in FIG. Figure 2 As shown, the advantageous 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-glufosinate were 277.8%, 250.1%, 300.7%, and 369.3% of that of the wild type, respectively, and the ee was ≥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 single-site saturation mutagenesis, the single mutants were subjected to iterative saturation mutagenesis. The primers are shown in Table 2. The enzyme activity of the transformants was tested using the method of Example 4. The results are shown in Table 2. Figure 3 As shown, the dominant 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. Figure 1In middle b, the relative activities of catalyzing the synthesis of L-glufosinate were 359.2%, 397.9% and 389.1% of the wild type, respectively, with ee ≥ 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, 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) inoculum amount, and cultured at 37 ° C, 180 rpm until the bacterial OD 600 When the pH reaches 0.4-0.6, add IPTG with an initial concentration of 0.1 M, induce culture at 28°C for 12 hours, centrifuge at 4°C and 8000 rpm for 10 minutes, 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: Detection of transaminase by SDS-PAGE electrophoresis

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

[0056] The wet cells of Example 1 E.coli BL21 (DE3) / pET28b-Se_WT and the dominant single mutant bacteria 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 with 20mM phosphate buffer (pH 8.0) and the cell concentration was adjusted to 10g / L. Ultrasonic disruption (40W, continuous disruption 1.5min) was performed in a low-temperature ice bath. After the disrupted cell solution was centrifuged at 12000rpm for 1min, the supernatant was the crude transaminase enzyme solution. The SDS-PAGE electrophoresis diagram 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 a theoretical molecular weight of 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 0.85% saline (NaCl), centrifuged at 4°C and 8000 rpm for 10 min, and the cells were collected again. The collected cells were resuspended in 20 mM phosphate buffer (pH 8.0) and the cell concentration was adjusted to 10 g / L. The cells were ultrasonically disrupted in an ice bath at low temperature (40W, 2s duration, 4s rest, 40min continuous disruption). The disrupted cell fluid was centrifuged at 12000 rpm for 10min, and 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 the crude enzyme solution is loaded 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 speed of 2mL / min for 5 column volumes and collect the eluate. Subsequently, the collected eluate is placed in a dialysis bag of 8000-14000Da size and dialyzed in a pH 8.0, 20mM phosphate buffer on ice for 12h for desalting. The buffer is changed every 6h. The retentate is the pure enzyme solution of the corresponding transaminase; after appropriate dilution, SDS-PAGE electrophoresis is performed, as shown in FIG. Figure 4 As shown in b, transaminase Se_WT and its mutants have a total of 435 amino acids (with 6×His-tag) and a theoretical molecular weight of 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 NaCl, 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 were used as catalyst, PPO as acceptor substrate, and L-alanine as donor for 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 1 U.

[0066] Relative enzyme activity: The relative enzyme activity of other mutant enzymes was calculated with the Se_WT enzyme activity as 100%.

[0067] The reaction system (1 mL) consisted of 50 mM PPO, 75 mM L-alanine, 0.1 mM PLP, and 10 g / L (WCW) bacterial cells in a Tris-HCl buffer (50 mM, pH 8.5) medium. The catalytic reaction was carried out at 35°C and 600 rpm. 200 μL of the sample was sampled periodically and terminated by adding 5 μL of 6 M HCl. The sample was centrifuged at 12,000 rpm for 1 min, and the supernatant was diluted appropriately. After derivatization, the peak area of ​​L-phosphinothricin was determined by high-performance liquid chromatography (HPLC). The L-phosphinothricin content of the product was calculated based on a standard curve of peak area and concentration of L-phosphinothricin standards under the same detection conditions, and the enzyme activity was then calculated.

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

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

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

[0071] The reaction system (10 mL) consisted of 50 mM PPO, 75 mM L-alanine, 0.1 mM PLP, and 10 g / L (WCW) bacterial cells in a Tris-HCl buffer (50 mM, pH 8.5) as the reaction medium. The catalytic reaction was carried out at 35°C and 600 rpm. 200 μL of the sample was taken at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h, and the reaction was terminated by adding 5 μL of 6 M HCl. The supernatant was then centrifuged at 12,000 rpm for 1 min, and diluted appropriately. After derivatization, the sample was detected using the method of Example 4. The results are shown in Table 4. Figure 5 .

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

[0073] Reaction equilibrium was achieved in 3-6 hours for Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D, respectively. At equilibrium, the yield of L-glufosinate was 41.5%, with product ee ≥99.9%. Compared to the wild-type transaminase Se_WT, the mutants Se_I22D, Se_N13M / I22D, Se_N13M / V19L / I22D, and Se_N13M / R17V / V19L / I22D exhibited a 12-15 hour reduction in reaction time and a 3-5-fold increase in space-time yield. These mutants offer promising applications for the asymmetric synthesis of L-glufosinate using L-alanine as an amino donor.

Claims

1. A transaminase mutant, characterized in that: The amino acid sequence of the transaminase mutant is mutated to one of the following: (1) asparagine at position 13 is substituted by methionine; (2) isoleucine at position 22 is substituted by aspartic acid and asparagine at position 13 is substituted by methionine; (3) 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; (4) 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.

2. A recombinant genetically engineered bacterium containing the gene encoding the transaminase mutant according to claim 1.

3. A transaminase mutant according to claim 1 in biocatalysis carbonyl 4 [Hydroxy(methyl)phosphono] Asymmetric synthesis of butyric acid Application of glufosinate ammonium.

4. The use according to claim 3, characterized in that The application is: using wet bacteria obtained by fermentation and culture of recombinant genetically engineered bacteria containing the transaminase mutant encoding gene or pure enzyme liquid obtained by ultrasonically crushing the wet bacteria and purifying it with a nickel column as a catalyst, carbonyl 4 [Hydroxy(methyl)phosphono] Butyrate is the substrate, pyridoxal phosphate is the coenzyme, and the natural amino acid L Alanine is used as an amino donor, and a conversion system is formed in a pH 7.0-9.0 buffer solution. The reaction is carried out at 30-50°C and 600-800 rpm. After the reaction is completed, the reaction solution is extracted to obtain L Glufosinate ammonium.

5. The use according to claim 4, 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; L The dosage of alanine is 50 mM to 150 mM.

6. The use according to claim 4, characterized in that The wet bacteria were prepared as follows: the engineered bacteria containing the transaminase mutant gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C and 200 rpm for 10-12 h, and then inoculated into 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 bacteria OD 600 When the p-value reaches 0.4-0.6, IPTG is added at a final concentration of 0.1 mM, and the culture is induced at 28°C for 12 h. The culture is then centrifuged at 4°C and 8000 rpm for 10 min, the supernatant is discarded, and the wet cells are collected. The TB culture medium is composed 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.

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