Transaminase mutants and uses thereof

Abstract

The application relates to application of a transaminase mutant in synthesis of a key chiral intermediate D-4,4'-diphenylalanine of sacubitril valsartan sodium salt (LCZ696), and belongs to the technical field of biocatalytic synthesis.The mutant is a quadruple mutant of the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of the transaminase mutant is shown in SEQ ID NO:3, and the corresponding amino acid sequence is shown in SEQ ID NO:4.Compared with a wild-type transaminase parent, the unit enzyme activity is increased by about 30 times under the condition of ensuring the chiral purity of a product, the transaminase has very high stereoselectivity and conversion rate, the industrial production cost of sacubitril and LCZ696 can be further reduced, and the transaminase has good industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biocatalytic synthesis technology, specifically, it relates to the application of a transaminase mutant in the synthesis of D-4,4'-biphenylalanine, a key chiral intermediate in sacubitril / valsartan sodium salt (LCZ696). Background Technology

[0002] LCZ696 (trade name Entresto, formula A) is a dual-action angiotensin receptor neprilysin (NEP) inhibitor developed by Novartis Pharmaceuticals in the United States. It consists of valsartan (formula B) and sacubitril (also known as AHU377, formula C) in a 1:1 molar ratio. It was approved by the FDA on July 7, 2015, for the treatment of hypertension and heart failure.

[0003]

[0004] Formula D (D-4,4'-biphenylalanine) is a key chiral intermediate in the preparation of sacurbitacin, and has become a critical factor restricting the production of sacurbitacin. Figure 1The chemical preparation process of Sakubiqu and its analogues has been reported in numerous patents (WO 2007 / 083774, WO 2007 / 083776, WO 2008 / 031567, WO 2008 / 083967, WO 2008 / 120567, WO 2009 / 090251, WO 2010 / 081410, WO 2011 / 035569, WO 2011 / 088797, WO 2012 / 025501, WO 2012 / 025502, WO 2013 / 026773, WO 2014 / 032627, WO 2015 / 024991, WO 2015 / 037460, CN101362708). (CN102260177, CN103483201, CN104557600, CN104725256, CN104725279, CN105017082, CN105061263, CN105085322, CN105152980, CN105168205, CN105198775, CN105237560, CN105330569, CN105481622, CN105566194, CN105601524 and CN105884656), but these methods still have obvious drawbacks, such as potentially hazardous reactants or the use of expensive catalysts and / or limited stereoselectivity. Therefore, there remains a need for better-designed preparation methods to provide an inexpensive way to obtain D-4,4'-biphenylalanine for synthesis, methods suitable for industrial-scale production under more economically and environmentally favorable conditions and providing D-4,4'-biphenylalanine with high chemical purity and high stereochemical selectivity.

[0005] WO 2018 / 116203 and CN 110088079 are used ( R A selective ω-transaminase catalyzed 4,4'-biphenylpyruvate (substrate) at 40-45℃ for 17-18 hours, achieving near 100% substrate conversion. Post-processing yielded D-4,4'-biphenylalanine with an ee value >99%, a yield of 90%. This demonstrates the significant advantages of enzymatic catalysis in the synthesis of this chiral compound. However, the long reaction time of this transaminase indicates room for improvement in its catalytic efficiency. Exploring transaminases from different biological sources and modifying enzyme molecules using protein engineering methods to obtain more efficient transaminases is a key direction for the industrial-scale enzymatic production of the crucial chiral intermediate D-4,4'-biphenylalanine from LCZ696. Summary of the Invention

[0006] Objective of the invention: To address the shortcomings of existing synthetic methods for preparing D-4,4'-biphenylalanine, this invention provides a preparation process suitable for green chemical industrial production, thereby further providing society with high-quality and low-cost raw materials.

[0007] Technical solution: This invention is based on screening... Mycolicibacteriumagri The strain's natural transaminase, and a transaminase mutant obtained through directed evolution based on this, were used to catalyze the synthesis of D-4,4'-biphenylalanine from 4,4'-biphenylpyruvate, as shown in the following reaction formula:

[0008]

[0009] In the formula: PLP is pyridoxal 5'-phosphate, which is a coenzyme for transaminase.

[0010] The present invention provides an improved transaminase, namely a transaminase mutant, which has significantly higher enzyme catalytic activity than wild-type transaminase.

[0011] The technical solution of the present invention is a transaminase mutant, which is a quadruple mutant of the amino acid sequence shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding coding gene is SEQ ID NO: 2), and the mutation sites include the following sites: R at position 60 is mutated to G, D at position 72 is mutated to A, K at position 196 is mutated to N, and Q at position 199 is mutated to L.

[0012] The amino acid sequence of the transaminase mutant of the present invention is shown in SEQ ID NO: 3, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO: 4.

[0013] According to another aspect of the present invention, a recombinant plasmid is provided, the recombinant plasmid containing the nucleotide sequence of any of the above-mentioned genes, further wherein the plasmid is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+ ), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C.

[0014] According to another aspect of the present invention, a host cell is provided, the host cell containing any of the above-mentioned recombinant plasmids, and the host cell includes a prokaryotic cell or a eukaryotic cell, wherein the prokaryotic cell is preferably an Escherichia coli BL21(DE3) cell.

[0015] According to another aspect of the invention, the use of a transaminase mutant in the preparation of D-4,4'-biphenylalanine is also provided, comprising: obtaining D-4,4'-biphenylalanine by asymmetric transamination using 4,4'-biphenylpyruvic acid as a substrate in the presence of the transaminase mutant.

[0016] Specifically, in the preparation of D-4,4'-biphenylalanine, the transaminase mutant described above is used to catalyze the transaminase reaction by controlling the pH in the range of 7.5-9.5 with a phosphate buffer solution, preferably 8.0-9.0. The pH remains basically stable during the reaction, and no additional acid or alkali solution adjustment is required.

[0017] During the reaction, if the pH is below 7.5 or above 9.5, the enzyme-catalyzed reaction rate will be significantly reduced or the substrate may not be able to react completely.

[0018] Specifically, in the preparation of D-4,4'-biphenylalanine, the transaminase mutant described above is used with the catalytic transaminase reaction temperature controlled between 25-45℃, preferably 30-40℃. If the reaction temperature is below 25℃ or above 45℃, the enzyme-catalyzed reaction rate will decrease, or even the substrate may not react completely. Attached image description:

[0019] Figure 1 Flowchart of chemical synthesis-transaminase-catalyzed preparation of sakubiqu.

[0020] Figure 2 Electrophoretic detection results of the transaminase mutant in preferred embodiment 6 of the present invention.

[0021] Figure 3 HPLC chromatogram of chiral purity of D-4,4'-biphenylalanine

[0022] Beneficial Effects: The technical solution of this invention involves mutating the parent gene of the wild-type transaminase shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding coding gene is shown in SEQ ID NO: 2) using a random mutation molecular biology method. This alters the amino acid sequence of the enzyme, resulting in changes to its structure and function. A tetrad mutant of the transaminase with the aforementioned mutation sites is then obtained through targeted screening. Using this transaminase mutant to catalyze the synthesis of D-4,4'-biphenylpyruvate, compared to the wild-type parent transaminase, the enzyme activity per unit is increased by approximately 30 times while maintaining the chiral purity of the product (Example 5). It exhibits very high stereoselectivity and conversion efficiency. Specifically, with 0.01 wt transaminase powder catalyzing 4,4'-biphenylpyruvate, the substrate conversion rate can reach 100% and the product ee value can reach 99.9% after 4-8 h of reaction. Compared with the existing optimal enzyme catalysis process (patent CN 110088079, enzyme catalysis reaction for 18 h, substrate conversion rate of 100%, ee value > 99%), this invention provides a new source of transaminase mutant and a technical solution with higher substrate conversion efficiency (under the condition of equivalent enzyme dosage, reaction for 4-8 h is higher than reaction for 17-18 h). It can further reduce the industrial production cost of sacubitril and LCZ696 and has good industrial application value.

[0023] Detailed Implementation: The present invention will be further described below with reference to specific implementation examples, but the scope of protection of the present invention is not limited thereto:

[0024] Example 1: Obtaining Mycolicibacteriumagri Wild-type transaminase parent recombinant plasmid of the strain

[0025] Obtained from the NCBI Protein database Mycolicibacteriumagri The amino acid sequence of the transaminase parent strain (NCBI Reference Sequence: WP_097938638.1, SEQ ID NO: 1) and gene sequence (NCBI Reference Sequence: NZ_PDCP01000006.1, base sequence positions 40721~41737, SEQ ID NO: 2) were codon-optimized and the full-length gene was artificially synthesized by a service provider into the pET28a(+) expression plasmid. This plasmid was then transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 mg / L kanamycin sulfate, and incubated overnight at 37°C. Several single colonies were selected and cultured overnight on LB medium (containing 50 mg / L kanamycin sulfate) at 37°C. Recombinant plasmids were then extracted using a plasmid miniprep kit, and verified by PCR and sequencing to obtain the recombinant plasmid of the wild-type transaminase parent strain.

[0026] Example 2: Random mutation of the maternal gene for wild-type transaminase

[0027] According to the content described in Example 1, with the following... Mycolicibacteriumagri Using the recombinant plasmid encoding the transaminase parent gene of the strain as a template, primers were designed and synthesized at both ends using Primer 5.0 based on the transaminase parent gene (Table 1). Error-prone PCR technology (materials and concentrations are shown in Table 2, reaction conditions in Table 3) was used to obtain a linear gene fragment containing numerous base mutations. These PCR products and the pET28a(+) expression plasmid were digested with enzymes, gel-extracted, ligated, and transformed into *E. coli* BL21(DE3) competent cells, respectively. The cells were plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C. See Tables 1-3 for details.

[0028]

[0029]

[0030] Example 3: Cloning and Expression of Transaminase Mutants

[0031] To facilitate the cloning, expression, and identification of transaminase mutants, compatible restriction endonuclease sites were designed at the 5' and 3' ends of their genes, allowing for the use of... Nco I and Xho I Restriction endonucleases were used to simultaneously digest the target gene and pET28a(+) (other expression plasmids that can express proteins in E. coli can also be used), and the DNA was recovered via gel digestion. The larger fragments of the recovered target gene and plasmid were ligated using T4 DNA ligase. The ligation product was transformed into E. coli BL21(DE3) competent cells, and the transformed competent cells were then plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C.

[0032] Single colonies grown on the above culture dishes were inoculated into LB broth containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C with shaking. The bacterial cells were collected for plasmid extraction, PCR identification, and double enzyme digestion identification. The correct recombinant plasmid was named pET28a(+)-AN, and the *E. coli* containing the correct recombinant plasmid were subsequently induced to express the plasmid. The bacterial culture was then transferred to 500 mL of LB broth containing 100 mg / L ampicillin and cultured at 37°C with shaking until OD... 600 When the concentration of the bacterial culture is 0.6 to 0.8, add IPTG to a final concentration of 0.05 to 0.5 mM. Induce expression at 22 to 25°C for 12 to 16 h. After that, take out the bacterial culture, centrifuge at 6000 × g for 20 min to collect the bacterial cells, and freeze at -20°C for later use.

[0033] Example 4: Initial screening of transaminase mutants

[0034] According to the descriptions in Examples 2 and 3, single colonies from the above-mentioned LB agar medium were picked and inoculated into 48-well plates. 1 mL of LB medium containing 50 mg / L kanamycin sulfate was added to each well before incubation. The plates were cultured at 37°C and 220 rpm for 3 h with shaking. Then, a certain amount of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.15 mM) was added, and the plates were induced at 25°C and 220 rpm for 15 h. The cells were collected by centrifugation at 6000 ×g for 20 min. After discarding the supernatant, the cells were resuspended in 0.5 mL of potassium phosphate buffer (100 mM, pH 8.5). 0.1 mL of 4,4'-biphenylpyruvate solution (2 mM), 0.2 mL of 70% isopropylamine solution, and 0.2 mL of PLP solution (2 mM) were added. The total reaction volume was 1.0 ml (mM), and the reaction was carried out at 40 °C for 22 h. The reaction was terminated by adding 1 mL of methanol. After shaking and centrifugation, the supernatant was collected and sent to HPLC for conversion analysis.

[0035] Example 5: Rescreening of transaminase mutants

[0036] (1) Preparation of transaminase mutant enzyme solution

[0037] The mutant strains with higher enzyme activity than the parent strain in Example 4 were inoculated at a rate of 0.1% into 10-20 bottles of 500 mL LB medium containing 100 mg / L ampicillin. The cultures were incubated at 37°C with shaking at 220 rpm for 5-6 h. A certain amount of the inducer isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.1 mM) was added, and the cultures were induced at 25°C with shaking at 220 rpm for 16 h. The cells were collected by centrifugation at 6000 ×g for 20 min. The cells were resuspended at 200 g / L whole cells using 100 mM potassium phosphate buffer (pH 8.5). The cell walls were ruptured using a high-pressure homogenizer (800-900 bar). The lysate was centrifuged at 4°C with centrifugation at 10000 ×g for 30 min to obtain the supernatant, which was the crude enzyme solution of the transaminase mutant.

[0038] (2) The reaction of 4,4'-biphenylpyruvic acid to D-4,4'-biphenylalanine catalyzed by transaminase

[0039] Add 0.2 g of the main raw material 4,4'-biphenylpyruvic acid, 1.0 g of dipotassium hydrogen phosphate trihydrate, 10 mL of 50 mM (pH 8.5) potassium phosphate buffer, 1.0 mL of 70% isopropylamine solution, 1.0 mL of 30% Tween-20 solution, and 0.4 mL of coenzyme pyridoxal phosphate solution (2 mM) to a 20 mL reaction flask to form a reaction system. Raise the temperature to 35 °C, add 50 mg (0.25 wt) of transaminase mutant enzyme solution, and adjust the reaction pH to 8.5-9.0 with 2 mol / L NaOH solution. Stop the reaction after 16 hours, and analyze the conversion rate and ee value by HPLC.

[0040] Comparative Example: 0.2 g of the main raw material 4,4'-biphenylpyruvic acid, 1.0 g of dipotassium hydrogen phosphate trihydrate, 10 mL of 50 mM (pH 8.5) potassium phosphate buffer, 1.0 mL of 70% isopropylamine solution, 1.0 mL of 30% Tween-20 solution, and 0.4 mL of coenzyme pyridoxal phosphate solution (2 mM) were added to two 20 mL reaction flasks respectively to form a reaction system. The temperature was raised to 35℃, and 50 mg (0.25 wt) and 500 mg (2.50 wt) of the transaminase parent enzyme solution were added respectively. The pH of the reaction was adjusted to 8.5-9.0 using 2 mol / L NaOH solution. The reaction was terminated after 16 hours, and the conversion rate and ee value were analyzed by HPLC.

[0041] (3) Determination of conversion rate in transaminase-catalyzed reaction

[0042] The reaction system was diluted with a solvent [0.01 mol / L potassium dihydrogen phosphate solution (containing 0.1% phosphoric acid) – acetonitrile (50:50)], filtered through a membrane, and then directly injected into the HPLC system for analysis. The HPLC conditions were as follows:

[0043] Instrument: Thermo U3000 system HPLC

[0044] Column: Agilent ZOBAX SB-phenyl, 4.6 mm × 250 mm, 5 μm

[0045] Mobile phase: 0.01 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B. Linear gradient elution was performed according to the table below.

[0046]

[0047] Detection wavelength: 255 nm;

[0048] Flow rate: 1.0 mL / min;

[0049] Column temperature: 30℃;

[0050] Injection volume: 10 μL.

[0051] Conversion rate calculation formula:

[0052]

[0053] In the formula, A(P) is the peak area of ​​D-4,4'-biphenylalanine;

[0054] A(S) represents the peak area of ​​the starting material 4,4'-biphenylpyruvic acid.

[0055] (4) Identification of optical purity of transaminase catalytic products

[0056] Take an appropriate amount of the product, dissolve and dilute it with a solvent by sonication to prepare a solution containing approximately 2 mg per 1 ml. Measure 1 ml and place it in a 5 ml centrifuge tube. Add 0.5 ml of borate buffer and 0.5 ml of FMOC-Cl solution in sequence, shake for 1 minute, and then add 50 μl of acetic acid and shake well.

[0057] The HPLC conditions are as follows:

[0058] Instrument: Thermo U3000 system HPLC

[0059] Column: Chiralcel OJ-RH, 4.6 mm × 150 mm, 5 μm

[0060] Mobile phase: 0.1% trifluoroacetic acid acetonitrile solution as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B.

[0061] Detection wavelength: 263 nm;

[0062] Flow rate: 0.7 mL / min;

[0063] Column temperature: 25℃;

[0064] Injection volume: 3 μL.

[0065] Formula for calculating the optical purity of R-type products:

[0066]

[0067] In the formula, A(R) is the peak area of ​​the target product D-4,4'-biphenylalanine;

[0068] A(S) represents the peak area of ​​the enantiomer L-4,4'-biphenylalanine.

[0069] Mutants with superior catalytic activity compared to the parent strain were selected for sequencing. Mutation sites were analyzed, and it was determined that the tetrad mutant R60G-D72A-K196N-Q199L (SEQ ID NO: 3, corresponding to the nucleotide sequence of the encoding gene is SEQ ID NO: 4) exhibited significantly higher catalytic activity than the parent strain in this scheme, without a decrease in the product ee value. The results of the secondary screening reaction are shown in Table 4.

[0070]

[0071] Note: Table 4 * This refers to the mass (g) of each transaminase solution required to transform 1 g of substrate. 0.25wt means that 0.25 g of transaminase mutant enzyme solution is required to transform 1 g of main raw material.

[0072] Table 4 shows that the catalytic efficiency of the R60G-D72A-K196N-Q199L quadruple mutant for 4,4'-biphenylpyruvate is about 30 times that of the wild-type transaminase parent, and the product has an ee value as high as 99.9% as the parent.

[0073] Example 6: Preparation of R60G-D72A-K196N-Q199L quadruple mutant enzyme powder

[0074] The mutant strain from Example 5 was inoculated at a rate of 0.01% into LB medium (500 mL / bottle * 30 bottles) containing 50 mg / L kanamycin sulfate and cultured at 37°C with shaking at 220 rpm for 5–6 h. A certain amount of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.15 mM) was added, and the culture was induced at 25°C with shaking at 220 rpm for 16 h. The cells were collected by centrifugation at 6000 ×g. The resulting 90–100 g of cells were resuspended in 200 mL of 50 mM potassium phosphate buffer (pH 8.5), homogenized, and then homogenized using a high-pressure homogenizer (800–900 bar). The supernatant was obtained by centrifugation at 4°C with 6000 ×g for 20 min. The SDS-PAGE spectrum of protein expression in the supernatant of the transaminase mutant is shown below. Figure 2 The supernatant was pre-frozen at -80℃ and then further freeze-dried in a SCIENTZ-25T freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.) to produce transaminase powder, which was then refrigerated at 2~8℃ for later use.

[0075] Example 7: Application of the tetratransaminase mutant in the preparation of D-4,4'-biphenylalanine

[0076] 10 g of 4,4'-biphenylpyruvate, 8 g of dipotassium hydrogen phosphate trihydrate, 50 g of 50 mmol / L (pH 8.5) potassium phosphate buffer, 3.51 g of 70% isopropylamine solution, 5 g of 30% Tween-20 solution, and 67 mg of pyridoxal coenzyme phosphate were mixed to form a reaction system. The temperature was raised to 35℃ under magnetic stirring, and 0.1 g (0.01 wt) of tetrahydrotransferase mutant enzyme powder was added. The pH of the reaction was adjusted to 8.5-9.0 using 2 mol / L NaOH solution. The reaction was monitored by HPLC until 6.5 h, when the residual 4,4'-biphenylpyruvate substrate was 0.08% (HPLC, peak surface normalization method), at which point the reaction was stopped. The reaction system was filtered, and the filter cake was washed successively with 50 mL of 50 mM (pH 8.5) potassium phosphate buffer and 50 mL of purified water. The filter cake was then heat-treated with 50 mL of methanol at 65°C for 1 hour, filtered and dried to obtain 9.34 g of product, with a yield of 93.1%, HPLC purity of 99.47%, and ee value of 99.82%.

[0077] Example 8: Scale-up application of the tetratransaminase mutant in the preparation of D-4,4'-biphenylalanine

[0078] Add 200.0 g of 4,4'-biphenylpyruvate, 160.1 g of dipotassium hydrogen phosphate trihydrate, 0.8 kg of 50 mmol / L (pH 8.5) potassium phosphate buffer, 50.0 g of isopropylamine, and 30.2 g of Tween-20 to a 3 L round-bottom three-necked flask. With mechanical stirring at 250 rpm, raise the temperature of the reaction system to 35 °C. Add 2.0 g (0.01 wt) of tetrahydrotransaminase mutant enzyme powder and 1.3 g of PLP monohydrate. Adjust the pH of the reaction to 8.5-9.0 using 2 mol / L NaOH solution. Monitor the reaction with HPLC until 8.0 h; when the substrate 4,4'-biphenylpyruvate is no longer detectable, stop the reaction. Filter the reaction system, and wash the filter cake successively with 500 mL of 50 mM (pH 8.5) potassium phosphate buffer and 500 mL of purified water. The filter cake was then heat-treated with 500 mL of methanol under reflux at 65°C for 1 hour. After cooling to room temperature, it was filtered. The resulting filter cake was dried at 60°C to obtain 95.4 g of product, with a yield of 95.2%. The HPLC-related purity of the product was 99.54%, and the ee value was 99.91%. Figure 3 ).

[0079] The results showed that the transaminase tetrapeptide mutant shown in SEQ ID NO:2 achieved 100% conversion and a product ee value of 99.9% in the enzyme-catalyzed 4,4'-biphenylpyruvate reaction system (i.e., 0.01 wt R60G-D72A-K196N-Q199L tetrapeptide mutant transaminase) after 8 h of reaction. The screened R60G-D72A-K196N-Q199L tetrapeptide transaminase mutant exhibited extremely high stereoselectivity and efficiency in the enzymatic preparation of D-4,4'-biphenylalanine.

Claims

1. A transaminase mutant, characterized in that, The amino acid sequence of the transaminase mutant is shown in SEQ ID NO:

3.

2. A recombinant plasmid, characterized in that, The plasmid contains the encoding gene of the transaminase mutant as described in claim 1.

3. The recombinant plasmid according to claim 2, characterized in that, The plasmid backbone of the stated plasmids is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+ ), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C.

4. A host cell, characterized in that, Contains the recombinant plasmid as described in claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is selected from prokaryotic or eukaryotic cells.

6. The host cell according to claim 5, characterized in that, The prokaryotic cells mentioned are Escherichia coli BL21(DE3) cells.

7. The application of the transaminase mutant according to claim 1, characterized in that, Application of catalytic synthesis of D-4,4'-diphenylalanine from 4,4'-biphenylpyruvic acid.

8. The application according to claim 7, characterized in that, The pH range of the catalytic reaction buffer solution is 7.5-9.5, and the temperature range is 25-45℃.

9. The application according to claim 8, characterized in that, The pH range of the catalytic reaction buffer solution is 8.0-9.0, and the temperature range is 30-40℃.

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