Amine dehydrogenase mutant and method for synthesizing non-α-amino acid

Abstract

This invention discloses a mutant amine dehydrogenase and a method for synthesizing non-α-amino acids, belonging to the field of biocatalysis engineering. This invention selects keto esters as substrates and utilizes phenylalanine amine dehydrogenase to complete the asymmetric reductive amination of these esters with different substitution positions. A strategy combining substrate engineering and enzyme evolution is employed to enhance substrate-enzyme compatibility, increasing the activity of β-, γ-, δ-, and ε-keto esters by 13-fold, 8-fold, 9-fold, and 40-fold, respectively. Results show that substrate engineering and enzyme evolution promote nonpolar interactions between the enzyme and substrate, improving substrate binding affinity. Using superior mutants, various non-α-amino acids were synthesized with high conversion rates and enantioselectivity exceeding 99%. The synthesized amino acids include β- and γ-amino acids, as well as δ- and ε-amino acids with more distant substitution positions. Therefore, an eco-friendly enzyme chemistry cascade has been successfully established for the synthesis of non-α-amino acids with different substitution patterns.

Description

Technical Field

[0001] The invention relates to a synthetic method of an amine dehydrogenase mutant and non-α-amino acid, and belongs to the field of biocatalytic engineering. Background Art

[0002] According to the different positions of the amino group on the carbon chain, amino acids can be divided into α-amino acids and non-α-amino acids. Chiral non-α-amino acids have important applications in biotechnology and fine chemicals. In the pharmaceutical field, chiral non-standard amino acids are the chiral cornerstones of best-selling drugs, such as (R)-3-aminobutyric acid in Durutevir and (R)-4-aminopentanoic acid in some neurological drugs ( Figure 1 A in ). In addition, non-α-amino acids participate in peptide chain folding, giving peptides functional properties such as inhibiting protein-protein interactions. The addition of non-α-amino acids to enzymes breaks the limitation that the genetic code can only contain 22 natural amino acids, so that biocatalysts with higher efficiency and specificity can be designed, and artificial enzymes can be created according to application requirements. Non-α-amino acids can be converted into lactams, such as pyrrolidine, which are key scaffolds and intermediates in drugs, dyes and inks, and can also be used as solvents and surfactants. The huge market demand highlights the importance of developing new synthetic pathways for chiral non-α-amino acids.

[0003] The chemical synthesis of chiral non-α-amino acids faces great challenges because of the need for precise chiral configuration at the carbon atom to which the amino group is attached. In addition, the amino and carboxyl groups are easily damaged during chemical synthesis, so they need to be protected. Traditional chemical synthesis of non-α-amino acids usually uses chemical or metal catalysts for reductive amination or asymmetric hydrogenation, which can be environmentally unfriendly and have inconsistent stereoselectivity. In recent years, significant achievements have been made in the enzymatic asymmetric synthesis of non-α-amino acids, providing a milder and more sustainable method compared to traditional chemical methods ( Figure 1B in the figure). Aminotransferase-mediated synthesis of β-amino acids, involving the transfer of an amino group to a β-keto acid carbonyl, is costly and inefficient due to equilibrium constraints and the need for an organic amine donor. Recently, we have successfully synthesized γ-amino acids through a substrate-specific modification strategy that involves altering the conversion-specific recognition group of the EcGluDH glutamate dehydrogenase. Current research has focused on the enzymatic synthesis of β- and γ-amino acids to facilitate the synthesis of non-α-amino acids. Nevertheless, substrate specificity remains a challenge for the biosynthesis of a variety of non-α-amino acids, especially for those with substituents far from the carboxyl group, such as δ- and ε-amino acids. In our previous study, EcGluDH was ineffective for the synthesis of δ-amino acids because the strong polarity of the carboxyl group hindered the generation of non-α-amino acids with different substitution sites. The hydrogen bonding interaction between K92, S378 and the carboxyl group prevented the key catalytic site from attacking the carbonyl group at different substitution positions. How to eliminate the specific recognition caused by this carboxyl group is the key to establishing a variety of non-α-amino acid synthesis pathways.

[0004] Substrate engineering is a key strategy to tune substrate-enzyme interactions. Previous studies have demonstrated its efficacy in expanding substrate scope and enhancing enantioselectivity. In organic synthesis, protecting groups such as Boc, Fmoc, and SEM are used to temporarily inhibit the reactivity of functional groups, and then they are removed after the reaction to produce the desired products. This concept can be transferred to enzyme synthesis, where non-polar group modification can mask the strong polar interactions between substrates and amino acid residues. This approach bypasses the positional restrictions of substrate substitution induced by carboxyl groups, and the target products can be subsequently obtained by simple chemical deprotection. Using ester groups to shield the polar effects induced by carboxyl groups is a key strategy in substrate engineering, so it is necessary to find enzymes that can catalyze the reductive amination of ketoesters.

[0005] Amine dehydrogenases (AmDHs), including engineered amine dehydrogenases obtained by directed evolution of widely used amino acid dehydrogenases and natural amine dehydrogenases, catalyze the synthesis of chiral amines via asymmetric reductive amination, using cost-effective ammonia donors to achieve high product conversion and enantioselectivity. Cascade catalytic systems containing AmDHs have been developed for the synthesis of chiral amines with excellent enantioselectivity. AmDH can catalyze small molecules with non-polar groups on the side chains. These studies include modifying the substrate binding site to accommodate larger non-natural substrates and expanding AmDH activity to include bicyclic and heterocyclic ketones. Currently, AmDHs show significant catalytic potential for substrates with non-polar side chains, meeting the requirements for catalyzing substrates modified with non-polar groups. Summary of the invention

[0006] In this study, a novel enzymatic chemistry cascade using AmDH was designed to synthesize non-α-amino acids with different substitution positions using ketoesters as substrates. Figure 1C in the figure). Two key elements to construct this cascade include removing the restriction on the substitution position and enhancing the catalytic activity of AmDH towards ketoesters. Based on substrate engineering, the carboxyl group was modified to an ester group to remove the restriction on the carbonyl substitution position. The combination of substrate engineering and steric modification of the enzyme effectively improved the catalytic activity of F-BbAmDH towards ketoesters. β-, γ-, δ-, and ε-amino acids were synthesized through a series of enzymatic reactions and chemical hydrolysis. The engineered mutants enrich the toolbox for the green synthesis of non-α-amino acids.

[0007] The present invention provides a phenylalanine amine dehydrogenase mutant, wherein the phenylalanine amine dehydrogenase mutant is obtained by mutating one or more amino acids at positions 50, 124, 144, 306, 309 and 313 of the phenylalanine amine dehydrogenase whose amino acid sequence is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] MSLVEKTSIIKDFTLFEKMSEHEQVVFCNDPATGLRAIIAIHDTTLGPALGGCRMQPYNSVEEALEDALRLSKGMTYSCAASDVDFGGGKAVIIGDPQKDKSPELFRAFGQFVDSLGGRFYTGTDMGTNMEDFIHAMKETNCIVGVPEAYGGGGDSSIPTAMGVLYGIKATNKMLFGKDDLGGVTYAIQG LGKVGYKVAEGLLEEGAHLFVTDINEQTLEAIQEKAKTTSGSVTVVASDEIYSQEADVFVPCAFGGVVNDETMKQFKVKAIAGSALNQLLTEDHGRHLADKGILYAPDYIVNSGGLIQVADELYEVNKERVLAKTKHIYDAILEVYQQAELDQITTMEAANRMCEQRMAARGRRNSFFTSSVKPKWDIRN

[0010] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme phenylalanine amine dehydrogenase is shown as SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012]

[0013] In one embodiment of the present invention, the phenylalanine amine dehydrogenase mutant is obtained by mutating the leucine at position 50 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and is named: L50A;

[0014] In one embodiment of the present invention, the threonine at position 124 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and is named: T124A;

[0015] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the valine at position 144 to alanine, which is named: V144A;

[0016] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the leucine at position 306 to alanine, which is named: L306A;

[0017] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the valine at position 309 to alanine, which is named: V309A;

[0018] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the leucine at position 313 to alanine, which is named: L313A;

[0019] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the valine at position 309 to alanine, and the leucine at position 50 to alanine, glycine or valine, and they are named as: V309A / L50A, V309A / L50G, V309A / L50V respectively;

[0020] In one embodiment of the present invention, the amino acid sequence of phenylalanine amine dehydrogenase as shown in SEQ ID NO.1 is obtained by mutating the valine at position 309 to alanine, and mutating the valine at position 144 to alanine or glycine, which are named as: V309A / V144A, V309A / V144G;

[0021] In one embodiment of the present invention, the valine at position 309 of the phenylalanine amine dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the leucine at position 306 is mutated to alanine, glycine or valine, and the resulting proteins are named: V309A / L306A, V309A / L306G, and V309A / L306V, respectively.

[0022] The present invention also provides a gene encoding the mutant.

[0023] The invention also provides a recombinant vector carrying the gene.

[0024] The present invention also provides a recombinant cell expressing the mutant or carrying the gene or the recombinant vector.

[0025] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0026] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned phenylalanine amine dehydrogenase mutant sequence, wherein the catalyst is any one of the following forms:

[0027] (1) culturing the recombinant expression transformant and isolating transformant cells containing the recombinase;

[0028] (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0029] (3) culturing the recombinant expression transformant, isolating transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a lyophilized enzyme powder.

[0030] The present invention also provides the use of the phenylalanine amine dehydrogenase mutant or the recombinant cell or the recombinant enzyme catalyst in catalyzing the reduction of potentially chiral ketoester compounds to prepare chiral non-α-amino acids.

[0031] In one embodiment of the present invention, the structural formula of the prochiral ketoester compound includes but is not limited to:

[0032]

[0033] In one embodiment of the present invention, the application is to add the phenylalanine amine dehydrogenase mutant or the recombinant cell or the recombinant enzyme catalyst to a medium containing glucose dehydrogenase, NAD +, a potential chiral ketoester compound, and glucose in a reaction system to prepare a chiral non-α-amino acid.

[0034] In one embodiment of the present invention, the reaction system further contains NH4OH / NH4Cl buffer.

[0035] In one embodiment of the present invention, the glucose dehydrogenase is derived from Bacillus amyloliquefaciens.

[0036] In one embodiment of the present invention, the application is: adding substrate ketoester (1c, 2d, 3b, 4b, 50 mM, the substrate is dissolved in NH 4 Cl / NH 4 OH buffer (pH 9.5, 3M)), glucose dehydrogenase, NAD + , glucose.

[0037] In one embodiment of the present invention, the recombinant mutant enzyme or the recombinant wild-type enzyme is prepared using 20 to 40 g / L -1 The BaGluDH enzyme is prepared by fermentation of recombinant cells containing F-BbAmDH and its mutants. -1 The method is prepared by fermentation of recombinant cells of BaGluDH, glucose (1.2-120 mM), substrate 1-100 mM, NH4OH / NH4Cl buffer (3 M; pH 7-9); 30-50°C, 0-300 rpm, 18-24 h.

[0038] In one embodiment of the present invention, glucose (75 mM), NAD+ (1 mM), NH4OH / NH4Cl buffer (3 M; pH 8.5); and the reaction system is incubated at 37°C and 200 rpm for 20 h.

[0039] The present invention also provides the use of the mutant, the gene, the recombinant vector, the recombinant cell or the method in the fields of food, biomaterials, medicine, additives or cosmetics.

[0040] Beneficial Effects

[0041] (1) We designed and developed an innovative enzymatic chemistry cascade to synthesize non-α-amino acids with different substitution sites using a strategic substrate engineering approach. The cascade utilizes cost-effective inorganic ammonia as an amine donor and produces only water, value-added gluconic acid, and easily recyclable alcohol as byproducts, thus providing a green and sustainable bioprocess.

[0042] (2) The cascade pathway can synthesize not only common β- and γ-amino acids, but also δ- and ε-amino acids with more distal substitution sites, a feat that was previously unattainable using biological methods.

[0043] (3) In order to effectively improve the activity of the enzyme, we performed steric engineering design on the enzyme and substrate to find the most suitable enzyme-substrate match. The activities of different substrates were 13 times, 8 times, 9 times, and 40 times of the initial level, respectively, proving the effectiveness of this strategy.

[0044] (4) Four non-α-amino acids with different substitution positions were synthesized via this cascade pathway with substrate conversion rates ranging from 30.26% to 96.58% and good stereoselectivity (>99% ee).

[0045] (5) This approach of combining substrate engineering with enzyme evolution can be applied to the synthesis of more non-α-amino acids and their derivatives with different substitution positions, providing a potential pathway for the biosynthesis of these chiral molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : A is the application of non-α-amino acids; B is the asymmetric synthesis of non-α-amino acids; C is the cascade designed in this study for the synthesis of non-α-amino acids.

[0047] Figure 2 : Structural model of F-BbAmDH and identification of mutation sites; wherein, A is the structure of the optional substrate; B is the structural overview of the complex of wild-type F-BbAmDH and NADH; C is the hydrogen bonding and hydrophobic interactions of the complex structural model of F-BbAmDH-1a; D is the screening of key mutagenesis sites.

[0048] Figure 3 : Specific activities of BbAmDH and engineered mutants towards substrates 1a-1c.

[0049] Figure 4 : A and B are BbAmDH and mutant T124A; C, E and F are representative frames in the dynamics simulation of three complex structures; D is the distance between 1a and the active site K90 and the coenzyme in the molecular dynamics simulation of BbAmDH-1a.

[0050] Figure 5 : A is the root mean square fluctuation value of 1b and 1c; B is the hydrophobic contact interaction score of 1a, 1b and 1c.

[0051] Figure 6: Expansion of the combinatorial mutagenesis strategy; A is a schematic diagram of the mutation sites and building blocks and the co-evolution strategy; B is the specific activity of F-BbAmDH and engineered mutants towards substrates 2b-4b.

[0052] Figure 7 : Enzyme chemical cascade reaction.

[0053] Figure 8 : SDS-PAGE profile of the purified enzyme. DETAILED DESCRIPTION

[0054] The chemicals used in this study are described in detail in the SI The following Examples Unless otherwise stated, all reagents and solvents were purchased from commercial sources and used as received.

[0055] The chemical reagents involved in the following embodiments are as follows:

[0056] Substrates: Except for ethyl acetoacetate, ketoesters (≥95%-99%) were purchased from Macklin, Sigma-Aldrich, Bideparmatech, Innochem and Aladdin. Chengdu Kamel Company was commissioned to synthesize ethyl acetyl valerate (≥90%). Standards: (R)-3-Aminobutyric acid (P1, ≥97%) was purchased from Macklin; 4-aminovaleric acid was extracted from Leyan; 5-aminohexanoic acid (P3) and 6-aminoheptanoic acid (P4) were synthesized in the laboratory. Others: acetonitrile (≥98%, Macklin), methanol (≥98%), methyl tert-butyl ether (MTBE) (≥98%, Macklin. All other chemicals were obtained from commercial sources of at least reagent grade and used without further purification.

[0057] The detection methods involved in the following embodiments are:

[0058] Determination of reductive amination activity (detection of phenylalanine amine dehydrogenase activity):

[0059] The reaction volume was 200 μL, 96-well plate, reaction system: 15 mM ketoester, 0.2 mM NADH, 3 M NH4Cl / NH4OH buffer (pH 9.5) and a certain amount of purified enzyme; reaction conditions: temperature was 37°C, reaction time was 25 minutes.

[0060] The utilization of NADH was detected by recording the decrease in absorbance at 340 nm using a MultiSkan GO UV spectrophotometer (Thermo Fisher Scientific).

[0061] The enzyme activity was defined as 1 unit (1 U) corresponding to the oxidation of 1 μM NADH per minute at 37°C.

[0062] Kinetic parameter determination method:

[0063] In 0.2 mM NADH, 3 M NH 4 The determination was performed in a ketoester concentration gradient from 0.1 to 80 mM (0.1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM) in the presence of Cl / NH4OH buffer (pH 9.5) and a certain amount of enzyme. The values ​​of Kcat and Km were obtained by fitting the initial velocity data to the Michaelis-Menten model with substrate concentration using nonlinear regression in GraphPad Prism 8 software (GraphPad software, La Jolla, CA, USA).

[0064] Chemical synthesis of amino acids as a standard

[0065] Some non-α-amino acid standards (P1 to P4, with specific structures shown in Table 7) were synthesized by chemical reduction amination of the corresponding keto acids using NaBH3CN, specifically:

[0066] Keto acid (2 mmol), NH4COOH (20 mmol), and NaBH3CN (4 mmol) were dissolved in methanol (20 mL) and separated using a 20*20 silica gel preparation plate with n-butanol / CH 3 COOH / H 2 O(8 / 1 / 1,v / v).

[0067] After that, the scraped silica gel was dissolved in a mixture of dichloromethane and methanol, and then filtered through filter paper using a vacuum pump to obtain a clear solution. The solvent was blown off with nitrogen, and the remaining solid was washed with 1 H-NMR was used as the standard for chiral analysis.

[0068] GC-FID and HPLC analysis

[0069] The conversion was determined by GC-fid analysis using nitrogen as carrier gas on a 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, USA). The conversion was determined using a Grace eco-cap EC-WAX gas chromatograph column (30 m×0.25 mm×0.25 μm). Analysis program: split ratio 30:1, pressure 90.0 kPa. Column temperature program: start at 90°C, keep warm for 2 minutes, 20°C·min-1 to 160°C, keep warm for 2 minutes, then 10°C·min-1 to 200°C, keep warm for 2 minutes.

[0070] The chirality of the product was determined by OPA / NAC derivatization technique. 10 mg NAC and 10 mg OPA were dissolved in 2.5 mL of derivatization buffer (including 2 ml of 0.2 M boric acid, pH 9.8, 0.5 mL of acetonitrile) to prepare the OPA / NAC derivatization agent. Chromatographic evaluation was performed using an Agilent-1260 HPLC system (Agilent Technologies Inc., Palo Alto, USA) equipped with a Hypersil ODS-2 reversed-phase column (250×4.6 mm, 2.5 μm). Pre-column sample automatic derivatization was performed. UV detection of the derivatized product was performed at 338 nm, 40 °C, and the mobile phase was 50 mM KH2PO4 (pH 8.0) / acetonitrile (83 / 17, v / v) at a flow rate of 1 mL min -1 .

[0071] The chemical structures of 1a (methyl acetoacetate), 1b (ethyl acetoacetate), 1c (propyl acetoacetate), 2a (methyl levulinate), 2b (ethyl levulinate), 2c (propyl levulinate), 2d (butyl levulinate), 3a (methyl acetobutyrate), 3b (ethyl acetobutyrate), 4a (methyl acetovalerate), and 4b (ethyl acetovalerate) involved in the following embodiments are as follows:

[0072]

[0073] For simplicity of presentation, the above compounds are represented by 1a, 1b, 1c, 2a, 2b, 2c, 2d, 3a, 3b, 4a, and 4b;

[0074] Meanwhile, the substrates 1a, 1b, 1c, 2a, 2b, 2c, 2d, 3a, 3b, and 4a were purchased from Macklin, Sigma-Aldrich, Bidepharmatech, Innochem and Aladdin, and 4b was purchased from Chengdu Kamel Pharmaceutical Co., Ltd. The substrates were prepared by pipetting the corresponding amount of substrates and dissolving them in 10 mL NH 4 Cl / NH 4 OH buffer (pH 9.5, 3 M).

[0075] Example 1: Screening of phenylalanine amine dehydrogenase

[0076] The specific steps are as follows:

[0077] The carboxyl group of the keto acid was modified into a methyl ester to verify whether AmDH could catalyze the keto ester substrate. Using acetoacetate and methyl acetoacetate as substrates, five laboratory-preserved amine dehydrogenases were tested: F-GkAmDH from kaustopilus Geobacillus (described in L.Liu, D.-H.Wang, F.-F.Chen, Z.-J.Zhang, Q.Chen, J.-H.Xu, Z.-L.Wang, G.-W.Zheng, Development of an engineered thermostable amine dehydrogenase for the synthesis of structurally diverse chiral amines, Catalysis Science&Technology 10(8)(2020)2353-2358), F-BbAmDH from badius Bacillus (SEQ ID NO.1), L-EsAmDH from sibiricum Exiguobacterium (described in F.-F.Chen, Y.-Y.Liu, G.-W.Zheng, J.-H.Xu, Asymmetric Amination of Secondary Alcohols by using a Redox-NeutralTwo-Enzyme Cascade, ChemCatChem 7(23)(2015)3838-3841.) and L-BcAmDH from Bacillus cereus (described in X.Mu, T.Wu, Y.Mao, Y.Zhao, Y.Xu, Y.Nie, Iterative Alanine ScanningMutagenesis Confers Aromatic Ketone Specificity and Activity of L-AmineDehydrogenases, ChemCatChem 13(24)(2021)5243-5253.) and cFLF-AmDH chimeric with F-BbAmDH and L-EsAmDH (described in J.Li, X.Mu, T.Wu, Y.Xu, High coenzyme affinity chimeric aminedehydrogenase based on domain engineering, Bioresour. Bioprocess. 9(1)(2022)33).

[0078] At 37°C, 15 mM substrate (acetoacetate or methyl acetoacetate, dissolved in 10 mL NH 4 Cl / NH 4 OH buffer (pH 9.5, 3M)) and 0.2mM NADH in NH4Cl / NH4OH buffer (3M, pH 9.5), react for 25min, use MultiSkan GO UV spectrophotometer (Thermo Fisher Scientific) to record the decrease in absorbance at 340nm, detect the utilization of NADH, and the enzyme activity is defined as 1 unit (1U) corresponding to the oxidation of 1μM NADH per minute at 37°C, and the activity of the above enzyme for acetoacetate or methyl acetoacetate is measured. The results are shown in Table 1, where ND means not detected. All determinations were performed in triplicate, and the values ​​are shown as mean ± standard deviation.

[0079] Table 1: Asymmetric reductive amination of β-keto acids and β-keto esters

[0080]

[0081] The results showed that only F-AmDH derived from phenylalanine dehydrogenase had activity towards β-ketoesters, while no amine dehydrogenase had activity towards β-ketoacids. The activities of F-GkAmDH and F-BbAmDH towards methyl acetoacetate were 0.41 mU·mg-1 and 1.05 mU·mg-1, respectively (Table 1).

[0082] In order to further improve the activity of phenylalanine dehydrogenase F-BbAmDH on acetyl A acid B ester (wherein A is B, C, D, E, and B is A, B, C, D), the present invention further transforms phenylalanine dehydrogenase F-BbAmDH.

[0083] Example 2: Design of mutants

[0084] The specific steps are as follows:

[0085] (1) Structural modeling

[0086] The structural models of F-BbAmDH and its mutants were constructed using the SWISS-MODEL server (https: / / swissmodel.expasy.org / ). The complex structural models of F-BbAmDH and its coenzyme NADH mutants were obtained by comparison with the reported homologous complex structure (PDB ID: 1C1D). The structures of the ligands were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The ligand energy was minimized using the OPTIMIZE plugin of PyMOL.

[0087] (2) Molecular docking and molecular dynamics simulation

[0088] Docking simulations were performed using AutoDock v.4.2 (Scripps Research Institute, California, USA). Before docking, each protein was prepared using a hydrogen protonation server at pH 9.5 to simulate the experimental reaction conditions. All water molecules were removed and nonpolar hydrogen atoms were added using MGLTools v.1.5.4. The components of the catalytic diamine (K90-D125) were defined as flexible residues. A 3D-spaced 1 / 4-kPa (2.3-kPa) was constructed around the active cavity of F-BbAmDH and its mutants. of The grid box was used as the search space for finding the appropriate substrate binding pose. The energy of the receptor-ligand interaction was calculated using MGLTools v.1.5.4. The docking pose with the lowest energy was selected for molecular dynamics simulation.

[0089] Molecular dynamics simulations were performed using the AMBER software package. The general AMBERff14SB force field was used for all molecular dynamics simulations. The protein was dissolved in a cubic box of TIP3P water molecules with an outer water layer thickness of more than . Then, sodium ions were added as counterions to the protein surface to construct a neutral simulation system. After appropriate parameterization and setup, the system was minimized to eliminate unfavorable contacts and allowed to relax. The system was then gradually heated from 0 to 303 K for 50 ps under constant material (N), volume (V) and temperature (T) (NVT ensemble). Subsequently, the system was dynamically re-equilibrated using a Langevin thermostat for 50 ps under the NPT ensemble of constant material (N), pressure (P) and temperature (T), with a collision frequency of 2 ps-1 and a pressure relaxation time of 1 ps. Unrestrained 100 ns production simulations were performed under NPT conditions and their convergence was evaluated by RMS deviation analysis. Simulation trajectory files were written every 5000 steps with an integration time step of 2 fs and then analyzed using the trajectory protocol. The features of the substrate binding pocket were determined by extracting 20 frames from the molecular dynamics simulation of the protein structure without substrate under stable RMSD and analyzed using the protein binding pocket computational tool at https: / / proteins.plus / . The RMSF of the substrate was analyzed using the last 1000 frames of the trajectory. The hydrophobic interactions between the protein and the substrate were analyzed from the 1000-frame structure, and the proportion of the time spent on the hydrophobic interactions between different residues and the substrate to the total simulation time was calculated to analyze the hydrophobic forces on the side chains.

[0090] (3) Selection of mutants

[0091] F-BbAmDH was selected as the mutagenesis template, and the crystal structure of the F-BbAmDH-NADH complex ( Figure 2 B) was obtained by AlphaFold2 modeling and comparison with the structure of a reported homologous complex (PDB ID: 1C1D). Docking of 1a with F-BbAmDH showed that although 1a forms hydrogen bonds with the carbonyl group through G52 and K90, the substrate is stabilized by nonpolar interactions ( Figure 2 C).

[0092] L50, M75, Y121, T122, T124, V144, S156, L306, V309 and L313 were selected as the final 10 candidate mutation sites ( Figure 2 D).

[0093] Example 3: Preparation of F-BbAmDH mutants

[0094] The specific steps are as follows:

[0095] 1. Preparation of recombinant vector pET-28a(+)-F-BbAmDH

[0096] The F-BbAmDH gene with a gene sequence as shown in SEQ ID NO.2 was chemically synthesized, and the pET-28a(+) plasmid was used as an expression vector. The F-BbAmDH gene was connected to the pET-28a(+) plasmid to prepare the recombinant vector pET-28a(+)-F-BbAmDH.

[0097] 2. Preparation and purification of mutants

[0098] (1) Using the recombinant vector pET-28a(+)-F-BbAmDH as a template, L50, M75, Y121, T122, T124, V144, S156, L306, V309 and L313 in the sequence of F-BbAmDH were mutated to alanine by whole plasmid PCR for targeted mutagenesis.

[0099] (2) The conditions for the whole plasmid PCR were 98°C for 1 min, (98°C for 30 sec, 55°C for 15 sec, 72°C for 2 min) × 29 cycles, 72°C for 10 min, and 16°C for 5 min. The PCR reaction tube contained 2 μL of forward and reverse primers (10 pM), 1 μL of template plasmid (10 ng), 25 μL of PrimeSTAR MAX DNA polymerase, and 22 μL of ultrapure H2O. The PCR product was analyzed by electrophoresis on an agarose gel and then purified using a PCR purification kit (Thermo Scientific).

[0100] The purified gene samples were digested with Dpn1 to remove the empty plasmid template. The enzyme digestion mixture was incubated at 37°C for 1 h and purified again using a PCR purification kit (Thermo Scientific). The DNA concentration of the purified samples was measured at 280 nm using a NanoDrop 2000c spectrophotometer (Thermo Scientific) and then transformed into Escherichia coli BL21 (DE3) cells;

[0101] The recombinant strains were prepared respectively: BL21 / pET-28a(+)-L50A, BL21 / pET-28a(+)-M75A, BL21 / pET-28a(+)-Y121A, BL21 / pET-28a(+)-T122A, BL21 / pET-28a(+)-T124A, BL21 / pET-28a(+)-V144A, BL21 / pET-28a(+)-S156A, BL21 / pET-28a(+)-L306A, BL21 / pET-28a(+)-V309A, BL21 / pET-28a(+)-L313A, and BL21 / pET-28a(+)-F-BbAmDH.

[0102] L50, M75, Y121, T122, T124, V144, S156, L306, V309 and L313 in the sequence of F-BbAmDH were mutated to alanine. The primer sequences involved are shown in Table 2:

[0103] Table 2: Primer sequences

[0104]

[0105] (3) Protein expression and purification

[0106] The recombinant cells obtained in step (2) were placed in a culture medium containing kanamycin (final concentration 50 mg L -1 ) in Luria Bertani medium and shake at 37°C and 200 rpm to OD600 = 0.6-0.8. Protein expression was induced with IPTG (final concentration = 0.1 mM), and the cells were shaken at 17°C and 200 rpm for 16 hours and used His-tagged proteins were purified using the ELISA purification system (GE Healthcare, Little Chalfont, UK).

[0107] Cells were resuspended with buffer A (50 mM potassium phosphate, 100 mM NaCl, and 20 mM imidazole; pH 7.5), followed by an ultrasonic cell disruption system (SCIENTZ-ID; Ningbo Science Biotechnology Co., Ltd., Ningbo, China), and then centrifuged at 12,000 rpm, 4°C for 30 min to remove cell debris and obtain a crude enzyme solution. The latter was passed through a 0.22 μm filter and loaded onto a standard Ni NTA affinity column (Thermo Fisher Scientific, Waltham, MA, USA) pre-equilibrated with buffer A and then eluted with a gradient of buffer B (50 mM potassium phosphate, 100 mM NaCl, and 500 mM imidazole; pH 7.5). The purity of the eluted fractions was determined by SDS-PAGE ( Figure 8 ).

[0108] Pure enzyme solutions containing the following mutants: L50A, M75A, Y121A, T122A, T124A, V144A, S156A, L306A, V309A, L313A, and pure enzyme solutions containing wild-type F-BbAmDH were prepared respectively.

[0109] (4) The activity of 1a, 1b, and 1c was evaluated using them as substrates. Subsequently, mutants with enhanced activity were successfully obtained for each substrate ( Figure 3 The specific reaction is: add 15mM substrate (the substrate is dissolved in 10mL NH 4 Cl / NH 4 The above pure enzyme solution was added to the reaction system of NH4Cl / NH4OH buffer (pH9.5, 3M) and 0.2mM NADH, respectively, and reacted in NH4Cl / NH4OH buffer (3M, pH 9.5) at 37°C for 25 minutes, and the change of NADH absorbance at 340nm was detected. The enzyme activity was defined as 1 unit (1U) corresponding to the oxidation of 1μM NADH per minute at 37°C, and the specific activity (enzyme activity / protein concentration, unit is mU / mg) was calculated. The value is the average of three separate measurements, and the results are shown in Table 3.

[0110] Table 3: Specific activity under different substrate conditions

[0111]

[0112] The results showed that for substrate 1a, the activity of mutant T124A (5.3 mU·mg-1) was higher than that of wild-type F-BbAmDH (1.05 mU·mg -1 ) was 5.0 times higher. In contrast, for 1b substrate, mutant T124A (6.21 mU mg -1 )、L306A(5.50mU·mg -1)and V309A (6.12 mU mg -1 ) were 2.4, 2.2 and 2.4 times that of F-BbAmDH, respectively. For 1c, V309A activity (14.57 mU·mg -1 ) increased by 14.3 times.

[0113] Example 4: Preparation of F-BbAmDH double mutant

[0114] In order to verify the effectiveness of this method, the catalytic activity of various ketoesters with replacement sites was improved by using this method, which promoted the synthesis of various non-α-amino acids. The mutant V309A was selected for further modification because its activity for all methyl ester-modified substrates was much higher than that of the wild type. The non-polar amino acid residues L50, V144 and L306 close to A309 at the bottom of the substrate binding pocket were selected for the next step of pocket remodeling. These amino acid residues are located near the ester group in the docking model of V309A and substrate molecules. We used hydrophobic amino acids with different spatial size characteristics such as leucine (L), valine (V), alanine (A) and glycine (G) as the final building blocks for mutagenesis to explore the spatial adaptability between substrate and enzyme ( Figure 6 A).

[0115] The specific steps are as follows:

[0116] (1) Construction of mutants

[0117] The construction of V309A / L50A, V309A / L50G, V309A / L50V, V309A / V144A, V309A / V144G, V309A / L306A, V309A / L306G, and V309A / L306V mutants was the same as in Example 3, using pET-28a(+)-V309A as a template to mutate the above sites. The primer sequences involved are shown in Table 4:

[0118] Table 4: Primer sequences for double mutations

[0119] Primer name sequence V309A / L50A-F Same as table 2 V309A / L50A-R Same as table 2 V309A / L50G-F CAGCAggaGGAGGATGTCGCATGCAGCCTTAC V309A / L50G-R ACATCCTCCtccTGCTGGGCCTAAAGTGGTATCA V309A / L50V-F CAGCAgttGGAGGATGTCGCATGCAGCCTTAC V309A / L50V-R ACATCCTCCaacTGCTGGGCCTAAAGTGGTATCA V309A / V144A-F Same as table 2 V309A / V144A-R Same as table 2 V309A / V144G-F ATTGCATCggcGGAGTACCGGAGGCTTATGGA V309A / V144G-R TACTCCgccGATGCAATTCGTCTCCTTCATTG V309A / L306A-F ATTCAGGAGGCgctATCCAAGCAGCTGACGAATTG V309A / L306A-R GATagcGCCTCCTGAATTAACAATGTAGTCGG V309A / L306G-F ATTCAGGAGGCggtATCCAAGCAGCTGACGAATTG V309A / L306G-R GATaccGCCTCCTGAATTAACAATGTAGTCGG V309A / L306V-F AGGAGGCgttATCCAAGCAGCTGACGAATTGT V309A / L306V-F CTTGGATaacGCCTCCTGAATTAACAATGTAGTCG

[0120] According to the method of Example 3, recombinant cells containing mutants were prepared: BL21 / pET-28a(+)-V309A / L50A, BL21 / pET-28a(+)-V309A / L50G, BL21 / pET-28a(+)-V309A / L50V, BL21 / pET-28a(+)-V309A / V144A, BL21 / pET-28a(+)-V309A / V144G, BL21 / pET-28a(+)-V309A / L306A, BL21 / pET-28a(+)-V309A / L306G, BL21 / pET-28a(+)-V309A / L306V.

[0121] At the same time, pure enzyme solutions containing the following mutants were prepared: V309A / L50A, V309A / L50G, V309A / L50V, V309A / V144A, V309A / V144G, V309A / L306A, V309A / L306G, and V309A / L306V.

[0122] (2) Activity detection of different substrates (2b, 2c, 2d, 3a, 3b, 4a, 4b). The specific reaction is: add 15mM substrate (substrate dissolved in 10mL NH 4 Cl / NH 4 Pure enzyme solution was added to the reaction system of NH4Cl / NH4OH buffer (pH 9.5, 3M) and 0.2mM NADH, respectively, and after reacting in NH4Cl / NH4OH buffer (3M, pH 9.5) at 37°C for 25 minutes, the absorbance change of NADH at 340nm detection wavelength was measured. Enzyme activity is defined as 1 unit (1U) corresponding to the oxidation of 1μM NADH per minute at 37°C, and the specific activity (enzyme activity / protein concentration, unit is mU / mg) is calculated. The values ​​are the average of three separate measurements, and the results are shown in Table 5.

[0123] Table 5: Specific activity under different substrate conditions (2b, 2c, 2d, 3a, 3b, 4a, 4b)

[0124]

[0125]

[0126] The results showed that the catalytic activity of V309A / L50A gradually increased with the extension of the γ-substrate ester group, and the activities of 2b, 2c and 2d were 7.84, 10.04 and 16.56 mU·mg-1, respectively. Its activity for 2d was nearly 20 times higher than that of F-BbAmDH. Compared with V309A, the activity of V309A / L50A for 3a increased slightly to 42.48 mU·mg-1, and the activity for 3b increased from 9.53 mU·mg-1 to 59.04 mU·mg-1. The activity of the ε-ketoester of V309A / V144G for 4b was 94.20 mU·mg-1, which was 7.2 times that of V309A. In summary, the activities of the four types of substrates with different substitution positions were greatly enhanced ( Figure 6 ).

[0127] Example 5: Apparent Kinetic Parameter Analysis

[0128] The kinetic parameters of F-BbAmDH and representative superior mutants of 1c, 2d, 3b and 4d were measured using pure enzymes to explore how pocket remodeling affects the catalytic efficiency of the enzyme (Table 6).

[0129] Kinetic parameters: Activity was determined at 37°C with 0.1-80 mM substrate and 0.2 mM NADH in NH4Cl / NH4OH buffer (3 M, pH 9.5). ND means not detected. All assays were performed in triplicate and values ​​are shown as mean ± standard deviation.

[0130] Table 6: Kinetic parameters of F-BbAmDH and representative dominant mutants for 1a, 2d, 3b and 4b

[0131]

[0132] The results show:

[0133] (1) F-BbAmDH has the highest catalytic efficiency (kcat / Km) for substrate 1c. When the substrate is extended to 4d, the catalytic activity of F-BbAmDH is lost. For substrate 1c, the catalytic efficiency of V309A is 4.22 times that of F-BbAmDH. For substrates 2d and 3b, the catalytic efficiency of V309A is 1.30 and 2.16 times that of F-BbAmDH, respectively, and the catalytic efficiency of V309A / L50A is 16.67 and 40.66 times that of F-BbAmDH, respectively. The catalytic efficiency of the double-point mutant is greatly improved. F-BbAmDH could not catalyze the largest substrate 4b, but V309A produced catalytic activity towards it with a kcat / Km of 3.0×10-2mM-1min-1. The kcat / Km of the double-point mutant V309A / V144G (39.6×10-2mM-1min-1) was 13.2 times higher than that of V309A.

[0134] (2) Compared with the change in Km values, the increase in kcat / Km values ​​for all substrates was mainly caused by the increase in the kcat values ​​of the mutants, which is similar to the results of previous studies by our group and other studies. In substrates 1c (1.06min-1), 2d (1.59min-1), and 3b (3.23min-1), the kcat of the optimal mutant increased significantly compared with the wild type, increasing by 3.58 times, 10.46 times, and 24.18 times, respectively, while the absolute value of kcat of the optimal mutant on substrate 4b was very high. The phenomenon of kcat leading to improved catalytic efficiency is more obvious in large substrates. Although the Km value did not change much, the overall trend was consistent with our conjecture. Pocket remodeling improves the spatial compatibility between the enzyme and the substrate, and as the mutation occurs, the enzyme's absorption rate of the substrate gradually increases as the Km decreases.

[0135] Example 6: Enzymatic cascade reaction for the synthesis of non-α-amino acids

[0136] The analytical-scale synthesis was carried out on a 1 mL scale by coupling F-BbAmDH or its engineered mutants with BaGluDH. The specific steps are as follows:

[0137] (1) Preparation of crude enzyme solution:

[0138] The recombinant cells containing F-BbAmDH and its mutants prepared in Example 3 and Example 4 and the cells expressing BaGluDH purchased from Sigma-Aldrich were placed in a culture medium containing kanamycin (final concentration 50 mg L -1 ) in LuriaBertani medium and shaken at 37°C and 200 rpm to OD600 = 0.6-0.8. Protein expression was induced with IPTG (final concentration = 0.1 mM), and after shaking the cells at 17°C and 200 rpm for 16 h, the cells were resuspended with NH4Cl / NH4OH buffer (3 M, Ph 8.5; pH 7.5), followed by ultrasonic cell disruption system (SCIENTZ-ID; Ningbo Science Biotechnology Co., Ltd., Ningbo, China), and then centrifuged at 12,000 rpm, 4°C for 30 min to remove cell debris and obtain crude enzyme solution.

[0139] (2) We added substrate and NAD to the AmDHs crude enzyme solution + , only mixed with BaGluDH crude extract in equal proportion to initiate the reaction. Specifically:

[0140] According to the method of step (1), the cell-free extract of Escherichia coli wet cells containing F-BbAmDH and its mutants prepared in Examples 3 and 4 (including 20 g L -1F-BbAmDH and its mutants, 24 g L -1 BaGluDH), and crude enzyme solutions of F-BbAmDH and its mutants V309A, V309A / L50A, V309A / L50A, V309A / V144G, and BaGluDH were prepared respectively.

[0141] Final reaction system: Add substrate ketoester (1c, 2d, 3b, 4b, 50 mM, substrate dissolved in 10 mL NH 4 Cl / NH 4 OH buffer (pH 9.5, 3 M), glucose (75 mM), NAD+ (1 mM), 1 mL NH4OH / NH4Cl buffer (3 M; pH 8.5); and incubating the reaction system at 37°C and 200 rpm for 20 h;

[0142] After the reaction was completed, part of the reaction sample was taken out, ethanol was added to terminate the reaction, and the conversion rate was detected by GC-FID.

[0143] (3) The remaining reaction mixture was added to a NaOH (10 M) solution, and the reaction was terminated by soaking in water at 60°C for 2 h. The intermediate product was hydrolyzed to obtain amino acids. The ee value of the reaction was determined by comparing with the standard by high performance liquid chromatography, and all products were roughly separated by thin layer chromatography and confirmed by LC-MS and 1H NMR. The results are shown in Table 7.

[0144] Table 7: Asymmetric reductive amination of 1c, 2d, 3b and 4b and enzymatic chemical cascade synthesis of β-, γ-, δ- and ε-amino acids (P1-P4)

[0145]

[0146] a: Conversion was determined by GC-FID analysis. b: ee values ​​were determined by chiral HPLC analysis of the hydrolyzate of the enzymatic reaction product, and the absolute configuration of the product was determined by comparing the optical rotation data with literature data.

[0147] The results showed that 50 mM substrates 1c, 2d, 3b and 4b were used for asymmetric reductive amination, and glucose dehydrogenase from Bacillusamyloliquefaciens (BaGluDH) was introduced for coenzyme regeneration. Substrates 1c, 3b and 4b were aminated by the corresponding engineered mutants with high conversion rates (66.7%-96.5%), while substrate 2d had a conversion rate of 30.26% due to the protein toxicity of the cosolvent DMSO (20%, v / v). In contrast, F-BbAmDH could not reduce the amination of substrate 4b, and the conversion rates of substrates 1c, 3b and 4b were lower than those of the mutants. After the amino acid esters were prepared by reductive amination, the enzymatic reaction products were hydrolyzed in NaOH solution to obtain non-α-amino acids, and their configurations were analyzed. These mutants had perfect stereoselectivity, and all non-α-amino acid products had up to 99% R configuration.

[0148] (4) Expanding the synthesis scale of (R)-3-aminobutyric acid

[0149] To verify the application potential of this cascade, the valuable non-α-amino acid (R)-3-aminobutyric acid (P1), an important chiral component of Dolutegravir, was synthesized on a 200 mL scale using mutant V309A.

[0150] The specific steps are as follows:

[0151] The reaction system contained 100 mM acetoacetate (1c), 1 mM NAD+, and the wet cell-free extract of Escherichia coli obtained in step (2) (20 g L -1 F-BbAmDH mutant V309A, 24 g L -1 BaGluDH), 75 mM glucose, 200 mL NH4OH / NH4Cl buffer (3 M, pH 8.5), and the reaction system was incubated at 37°C and shaken at 200 rpm for 20 h.

[0152] During the reaction, the pH value of the mixture was maintained at 8.5 by titrating concentrated NH3·H2O. Subsequently, 10M NaOH was added and the ester group was removed by water bath at 60°C for 2 h. After removing the precipitate, (R)-3-aminobutyric acid (P1) in the solution was detected by HPLC (same as above), and the yield was calculated by comparison with the standard curve.

[0153] The results showed that the conversion rate of product P1 was 93.24% and the yield was 86.2%. These results demonstrate the potential of using the mutant as a biocatalyst to synthesize pharmaceutically relevant non-α-amino acids.

[0154] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A phenylalanine amine dehydrogenase mutant, characterized in that: The phenylalanine amine dehydrogenase mutant is, The amino acid sequence of phenylalanine amine dehydrogenase shown in SEQ ID NO. 1 is obtained by mutating the valine at position 309 to alanine, and mutating the leucine at position 50 to alanine or glycine; or the amino acid sequence of the phenylalanine amine dehydrogenase shown in SEQ ID NO.1 is obtained by mutating the valine at position 309 to alanine, and mutating the valine at position 144 to alanine or glycine; Or the amino acid sequence of the phenylalanine amine dehydrogenase shown in SEQ ID NO.1 is obtained by mutating the valine at position 309 to alanine, and mutating the leucine at position 306 to alanine or glycine.

2. A gene encoding the mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or the recombinant vector according to claim 2.

4. The recombinant cell according to claim 3, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

5. A recombinant enzyme catalyst comprising the phenylalanine amine dehydrogenase mutant sequence of claim 1, characterized in that: Is any of the following: (1) culturing a recombinant expression transformant, isolating a transformant cell expressing a recombinant enzyme containing the phenylalanine amine dehydrogenase mutant sequence of claim 1, and obtaining the recombinant enzyme catalyst; (2) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the phenylalanine amine dehydrogenase mutant sequence of claim 1, disrupting the transformant cells expressing the recombinant enzyme to obtain a cell disrupted liquid, and obtaining the recombinant enzyme catalyst; (3) Cultivating the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the phenylalanine amine dehydrogenase mutant sequence of claim 1, disrupting the transformant cells expressing the recombinant enzyme to obtain a cell disruption solution, and freeze-drying the cell disruption solution to obtain a freeze-dried enzyme powder to obtain the recombinant enzyme catalyst.

6. A method for increasing the activity of phenylalanine amine dehydrogenase on a substrate, characterized in that: The method comprises the following steps: mutating the leucine at position 309 of the phenylalanine amine dehydrogenase of the amino acid sequence as shown in SEQ ID NO.1 to alanine, and the substrate is any one of methyl acetoacetate, ethyl acetoacetate, propyl acetoacetate, methyl levulinate, ethyl levulinate, propyl levulinate, butyl levulinate, methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate and ethyl acetovalerate; or the valine at position 309 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the leucine at position 50 is mutated to alanine, and the substrate is any one of methyl levulinate, ethyl levulinate, propyl levulinate, butyl levulinate, methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate, and ethyl acetovalerate; Or the valine at position 309 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the leucine at position 50 is mutated to glycine, and the substrate is any one of ethyl levulinate, propyl levulinate, butyl levulinate, methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate, and ethyl acetovalerate; or the valine at position 309 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the valine at position 144 is mutated to alanine or glycine, and the substrate is any one of methyl levulinate, ethyl levulinate, propyl levulinate, butyl levulinate, methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate, and ethyl acetovalerate; Or the valine at position 309 of the phenylalanine amine dehydrogenase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the leucine at position 306 is mutated to alanine, and the substrate is any one of methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate, and ethyl acetovalerate; Or the valine at position 309 of the phenylalanine amine dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the leucine at position 306 is mutated to glycine, and the substrate is any one of propyl levulinate, methyl acetobutyrate, ethyl acetobutyrate, methyl acetovalerate, and ethyl acetovalerate.

7. Use of the phenylalanine amine dehydrogenase mutant according to claim 1, the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5 in catalyzing the reduction of prochiral ketoester compounds to prepare chiral non-α-amino acids.

8. The use according to claim 7, characterized in that: The structural formula of the potential chiral ketoester compound is:

9. The use according to claim 7, characterized in that: The application is to add the phenylalanine amine dehydrogenase mutant or the recombinant cell or the recombinant enzyme catalyst to a cell containing glucose dehydrogenase, NAD + , a potential chiral ketoester compound, and glucose in a reaction system to prepare a chiral non-α-amino acid.

10. The use according to claim 9, characterized in that: The reaction system also contains NH4OH / NH4Cl buffer.

11. The use according to claim 9, characterized in that: The glucose dehydrogenase is derived from Bacillus amyloliquefaciens.

12. Use of the mutant according to claim 1, the gene or recombinant vector according to claim 2, the recombinant cell according to claim 3 or 4, the recombinase catalyst according to claim 5, the method according to claim 6, or any one of claims 7 to 11 in the fields of food, biomaterials, medicine, additives or cosmetics.

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