Imine reductase or its mutant and its application in catalytic asymmetric reduction to prepare α-arylacetamide and its derivatives

Abstract

The present invention provides an imine reductase mutant, which is a mutant obtained by mutation of imine reductase IR-117 derived from Sinorhizobium as a parent, wherein the mutant has the following sequence or an amino acid sequence with at least 99% identity to the following sequence and having imine reductase activity: the cysteine ​​at position 192 of the parent is mutated to glycine to obtain mutant IR-117-C192G; the amino acid sequence of imine reductase IR-117 is shown in SEQ ID No. 1. The present invention also provides the use of imine reductase IR-117 and imine reductase mutants in catalytic asymmetric reduction to prepare α-arylacetamides and their derivatives. The present invention promotes the tautomerism of substrate enamine-imine through the active center of the imine reductase, and can, under aerobic conditions, cooperate with the NADPH-mediated hydrogen transfer process to achieve asymmetric hydrogenation of enamides with high selectivity, providing a more economical and green application solution for obtaining high-value chiral fatty amines.

Description

Technical Field

[0001] The invention relates to an imine reductase or a mutant thereof and application thereof in catalytic asymmetric reduction to prepare alpha-arylacetamide and derivatives thereof, belonging to the field of enzyme engineering. Background Art

[0002] Chiral amine molecular skeletons are widely present in the fields of medicine, fine chemicals, and agriculture. As efficient stereochemical additives and synthetic building blocks, they have important research and application value. At present, the main strategy for the industrial production of optically pure amines is to use asymmetric hydrogenation reactions, using chiral catalysts as chiral sources to reduce unsaturated compounds such as chiral alkenes, ketones, enamines, and imines to prepare chiral amine products with diverse structures. Asymmetric hydrogenation of enamides has become one of the preferred methods for constructing multi-stereocentric chiral amines due to its high efficiency, ease of operation, simple post-processing, and ease of industrial scale-up (Synthesis, 2021, 53, 193-214).

[0003] Currently, the asymmetric hydrogenation of eneamides catalyzed by transition metals such as rhodium, rubidium, and iridium is the primary method for achieving asymmetric reduction of various eneamide substrates. Since the Kagan and Dang groups reported the first asymmetric hydrogenation of α-aryl eneamides catalyzed by a Rh(I)-DIOP complex in 1972 (J. Am. Chem. Soc, 2002, 94, 6429-6433), research on this reaction has been widely applied in the construction of macromolecular backbones for various pharmaceuticals. Zhang Xumu's group reported a novel DIOP ligand for rhodium-catalyzed asymmetric hydrogenation of eneamides, which exhibits extremely high reactivity and a broad substrate spectrum (J. Org. Chem, 2000, 65, 5871). However, this process requires a high rhodium catalyst loading (2% mol) and requires 10 atm H2 to maintain excellent stereoselectivity. Subsequently, Zhou Qilin et al. further developed a new monodentate phosphinamide ligand SiPhos, which achieved efficient asymmetric reduction of α-aryl olefin amides and their derivatives, with ee values ​​and yields greater than 99% (Angew. Chem. Int. Ed, 2002, 41, 2348). However, the reaction conditions are harsh, requiring toluene as the reaction solvent, and the hydrogen loading was increased to 50 atm at 5°C to maintain high yields and selectivities. In summary, transition metal-catalyzed asymmetric hydrogenation of olefin amides generally suffers from harsh reaction conditions, the need for the participation of noble metals and complex chiral ligands, the high environmental cost of using organic solvents, and metal residue problems, which are factors that are not conducive to large-scale industrial production.

[0004] With the development of biotechnology, biocatalytic reactions, with their advantages of mild reaction conditions, high specificity, and high selectivity, have gradually become a new and efficient way to obtain chiral molecular skeletons. In 2023, Huang Xiaoqiang's research group reported a new biosynthetic pathway for the preparation of N-acetyl-α-arylethylamine derivatives via photoenzyme-catalyzed asymmetric reduction, with high yields and a broad substrate spectrum (ACS Catal, 2023, 13, 15682-15690). However, this process requires visible light excitation and strict oxygen-free environment control, which increases the complexity of the process operation and limits its potential for further industrial application. Summary of the Invention

[0005] The present invention provides an imine reductase or a mutant thereof and application thereof in catalyzing asymmetric reduction to prepare α-arylacetamide and derivatives thereof.

[0006] Imine reductase is an NADPH-dependent oxidoreductase that catalyzes the asymmetric hydrogenation of the potentially chiral C=N bond of imines, particularly cyclic imines, to form corresponding chiral amine compounds. The present invention is based on the enzymatic enamine-imine tautomerism and hydrogen transfer mechanism, using α-phenylenamide as a substrate and imine reductase as a biocatalyst as a reaction pathway for the asymmetric reduction of α-arylacetamides and their derivatives. A candidate enzyme library is constructed, and imine reductases are screened using model reactions to obtain enzymes with initial reactivity. A mutant library with different amino acid residues is constructed, and the mutants are screened for reactivity and selectivity using directed evolution. Advantageous mutants with high reactivity and selectivity are obtained, and the applicable range of their reaction substrates is investigated.

[0007] The present invention provides an imine reductase mutant, which is obtained by mutating the imine reductase IR-117 from Sinorhizobium as a parent. The mutant has the following sequence or an amino acid sequence that is at least 99% identical to the following sequence and has imine reductase activity:

[0008] The cysteine ​​at position 192 of the parent was mutated to glycine to obtain the mutant IR-117-C192G.

[0009] The amino acid sequence of imine reductase IR-117 is shown in SEQ ID No. 1.

[0010] Furthermore, it is a mutant obtained by mutation of IR-117-C192G as a parent, wherein the mutant has the following sequence or an amino acid sequence having at least 99% identity with the following sequence and having imine reductase activity:

[0011] The asparagine at position 65 of the parent was mutated to serine to obtain the mutant IR-117-C192G-N65S.

[0012] Preferably, it is a mutant obtained by mutation of IR-117-C192G-N65S as a parent, wherein the mutant has the following sequence or an amino acid sequence having at least 99% identity with the following sequence and having imine reductase activity:

[0013] The glutamine at position 73 of the parent was mutated to alanine to obtain the mutant IR-117-C192G-N65S-Q73A.

[0014] Preferably, it is a mutant obtained by mutation of IR-117-C192G-N65S-Q73A as a parent, wherein the mutant has the following sequence or an amino acid sequence having at least 99% identity with the following sequence and having imine reductase activity:

[0015] The leucine at position 216 of the parent was mutated to methionine to obtain the mutant IR-117-C192G-N65S-Q73A-L216M.

[0016] More preferably, it is a mutant obtained by mutation of IR-117-C192G-N65S-Q73A-L216M as a parent, wherein the mutant has the following sequence or an amino acid sequence having at least 99% identity with the following sequence and having imine reductase activity:

[0017] The leucine at position 170 of the parent was mutated to phenylalanine to obtain the mutant IR-117-C192G-N65S-Q73A-L216M-L170F, whose amino acid sequence is shown in SEQ ID No. 2.

[0018] The present invention provides a DNA molecule, which encodes the imine reductase mutant.

[0019] The present invention provides an expression vector containing the DNA molecule.

[0020] The present invention provides a host cell, which contains the expression vector.

[0021] The present invention also provides use of imine reductase IR-117, the imine reductase mutant, the DNA molecule, the expression vector, or the host cell in catalyzing asymmetric reduction to prepare α-arylacetamide and its derivatives;

[0022] The α-arylacetamide and its derivatives are shown in Formula II and Formula IV:

[0023]

[0024] wherein R is selected from phenyl, C1-C6 substituted phenyl, heterocyclic substituent or C1-C6 straight chain or branched alkyl; R1 is independently selected from hydrogen or phenyl; R2 is independently selected from hydrogen or halogen atom; n=1,2;

[0025] Furthermore, the C1-C6 substituted phenyl group is selected from a halogen atom and a methyl group;

[0026] Furthermore, the heterocyclic substituent is selected from common heterocyclic structures such as thiophene.

[0027] The present invention provides a method for preparing α-arylacetamide and its derivatives by catalytic asymmetric reduction. The method uses formula I as a substrate and imine reductase IR-117, the imine reductase mutant, the DNA molecule, the expression vector, or the host cell as a catalyst to synthesize α-arylacetamide and its derivatives. The synthesis route is as follows:

[0028]

[0029] wherein R is selected from phenyl, C1-C6 substituted phenyl, heterocyclic substituent or C1-C6 straight chain or branched alkyl; R1 is independently selected from hydrogen or phenyl; R2 is independently selected from hydrogen or halogen atom; n=1,2;

[0030] Furthermore, the C1-C6 substituted phenyl group is selected from a halogen atom and a methyl group;

[0031] Furthermore, the heterocyclic substituent is selected from common heterocyclic structures such as thiophene;

[0032] Preferably, the reaction conditions are as follows: the reaction temperature is 30° C., the reaction time is 72 h, and the pH value is 6.0.

[0033] The present invention promotes substrate enamine-imine tautomerism through the active center of imine reductase, and can cooperate with the NADPH-mediated hydrogen transfer process under aerobic conditions to achieve asymmetric hydrogenation of enamides with high selectivity, providing a more economical and green application solution for obtaining high-value chiral fatty amines. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Recombinant plasmid map of IR117;

[0035] Figure 2 Recombinant plasmid map of GDH;

[0036] Figure 3 Standard curve of the product P3 corresponding to the model substrate S3 detected by LC-MS;

[0037] Figure 4 Figure 3. SDS-PAGE detection of mutant V5 purified protein (Note: M represents Marker; V5 supernatant represents V5 bacterial supernatant; Flow-through represents flow-through; numbers 1-5 represent purified mutant V5 protein);

[0038] Figure 5 The chiral detection original spectrum of the product P3 generated by the S3 reaction catalyzed by mutant V5 under optimal conditions (wherein, A is the chiral detection original spectrum of the racemate P3; B is the chiral detection original spectrum of the product P3: >99% ee generated by the S3 reaction catalyzed by mutant V5). DETAILED DESCRIPTION

[0039] Example 1: Construction of Imine Reductase and Investigation of Initial Reaction Activity

[0040] 1. Construction and cultivation of imine reductase pIR117 expression strain

[0041] Based on the design idea that the proton donor in the enzyme active center promotes the substrate enamine-imine tautomerism, a ligand-based There are acidic residues available in the range (as potential pIR117 (hereinafter referred to as IR-117) is a wild-type imine reductase from Sinorhizobium. First, IR-117 was codon-optimized, fully synthesized, and constructed using E. coli as the host cell to obtain the wild-type IR-117 recombinant expression vector pET28a-IR-117 (vector map shown in Figure 2). Figure 1 The expression vector was transformed into TSR2566 competent cells and cultured in an LB solid medium containing kanamycin at 37° C. for 12-16 h.

[0042] 2. Protein expression of imine reductase and preparation of crude enzyme solution

[0043] The successfully constructed IR-117 strain was inoculated into 3 mL of LB-Kan liquid medium and activated overnight at 37°C and 250 rpm. 1 mL of the activated bacterial solution was inoculated into 50 mL of TB-Kan liquid medium and cultured at 37°C and 220 rpm for 3 h. 600When the concentration reaches 0.6-0.8, place in a 4℃ refrigerator ice bath for 30 minutes, then add IPTG with a final concentration of 0.5mM and induce expression at 22℃, 150rpm for 18 hours. Centrifuge the induced expression solution to enrich the bacteria, discard the supernatant, and resuspend the residual precipitate in potassium phosphate (KPi, pH=7.0, 100mM) buffer, vortex mix and calculate the OD 600 Adjust to 30 as crude enzyme solution.

[0044] 3. Construction of glucose dehydrogenase (GDH) expression strain, protein expression and crude enzyme solution preparation

[0045] For glucose dehydrogenase (GDH, Genebank accession number: WP_003246720.1) from Bacillus subtilis, codon optimization, whole gene synthesis and vector construction were performed using Escherichia coli as the host cell to obtain the recombinant expression vector pET15b-GDH containing the glucose dehydrogenase gene (vector map as shown in Figure 2 The expression vector was transformed into TSR2566 E. coli competent cells, spread on LB-Amp solid medium and cultured inverted at 37°C for 12-16 hours to construct a GDH expression strain. The protein expression method was the same as in Example 1-2 (the culture medium antibiotic was Amp). 600 = 30, and crushed under pressure using a high-pressure homogenizer at 4°C and 800 bar for 30 minutes to prepare a crude GDH enzyme solution.

[0046] 4. Reactivity investigation

[0047] The wild-type imine reductase IR-117 constructed and expressed earlier was used to test the initial reaction activity of the model substrate α-phenylene amide (hereinafter referred to as S3). The specific operation is as follows: 200 μL of the prepared IRED crude enzyme solution, 200 μL of the GDH crude enzyme solution, 80 μL of NADP + A mixture of sodium salt (1.875mM, KPi solution) and D-Glucose (187.5mM, KPi solution) and 40μL substrate S3 (15mM, DMSO solution) were added to a 2mL reaction bottle and shaken at 25°C and 800rpm for 24h. After the reaction, 1400μL of anhydrous ethanol solution was added, shaken and mixed, and then centrifuged. 200μL of supernatant was aspirated into a 96-well ELISA plate, and the product concentration and chirality were detected by LC-MS. The product analytical yield was calculated by LC-MS detection of standards containing internal standards at different concentration gradients, and the peak area ratio of the standard to the internal standard was used to prepare a standard curve for calculation (hereinafter referred to as analytical yield). The standard curve is attached. Figure 3The chirality calculation is expressed as the ee value, ee = ((S, SR, R) / (S, S + R, R)) * 100%. The initial activity screening results are shown in Table 1.

[0048] Table 1 Initial activity screening results for model substrate S3

[0049]

[0050] Example 2: Directed evolution using wild-type IR-117 as a parent and model substrate S3 1. Construction of IR-117 mutant library

[0051] Using IR-117 as the parent, site-saturation mutagenesis was performed on S237, A208, and C192. First, the pET28a-IR-117 plasmid was used as the template and the corresponding degenerate primers were designed using the 22codon trick method for PCR reaction. The PCR reaction was performed using the high-fidelity DNA polymerase KOD One TM PCR Master Mix (TOYOBO) was used. The PCR reaction system is shown in Table 2:

[0052] Table 2 PCR reaction system

[0053]

[0054] PCR reaction conditions are shown in Table 3:

[0055] Table 3 PCR reaction conditions

[0056]

[0057] The linear DNA products obtained by PCR were subjected to 1% agarose gel electrophoresis and then recovered by gel cutting. The nucleic acid concentration of the gel recovered products was determined by ultra-micro nucleic acid analyzer and the DNA seamless cloning kit ( II One Step Cloning Kit, Novozymes) was used for ligation, and the ligation system is shown in Table 4:

[0058] Table 4 PCR product ligation system

[0059]

[0060] 2. Reactivity Screening

[0061] (1) Incubate the ligation system at 37°C for 30 minutes, take the incubation product and transform it into TSR2566 competent cells, and place it in a 37°C incubator for inversion culture for 12-16 hours. Pick a single colony on the plate and inoculate it into a 96-well plate containing 300μL LB-Kan liquid medium. Activate it in a shaker at 37°C and 250rpm overnight. Pipette 50μL of the activated bacterial solution and transfer it to a 96-deep-well plate containing 900μL TB-Kan liquid medium. Incubate it in a shaker at 37°C and 250rpm for 3 hours. Then, cool the 96-well plate in an ice bath for 30 minutes, add 50μL IPTG mother solution (10mM) to induce expression, and incubate it at 22°C and 200rpm for 18 hours. Enrich the bacteria by low-temperature centrifugation, add 100μL potassium phosphate buffer (pH 7.0, 100mM) with a pipette, and vortex to resuspend the bacteria. Then add 100 μL of the prepared GDH crude enzyme solution and 40 μL of NADP + A mixture of sodium salt (1.875 mM, KPi solution) and D-glucose (187.5 mM, KPi solution) was added to 20 μL of substrate S3 (15 mM, DMSO solution). The reaction mixture was sealed and incubated at 25°C with shaking at 800 rpm for 24 hours. After completion of the reaction, 900 μL of anhydrous ethanol was added, and the reaction activity was determined by LC-MS.

[0062] Mutants with enhanced activity initially detected in 96-well plates were cultured in shake flasks and their reaction activity was verified. The screening and detection methods for reaction activity verification were the same as those in Examples 1-4. The relative activity of the substrate S3 reaction was determined (relative activity was determined based on a curve of the corresponding peak area and product concentration measured by LC-MS; relative activity = mutant product concentration / parent product concentration in this round). The results of the first round of directed evolution verification are shown in Table 5.

[0063] Because the EE value of IR-117 in the initial screening results was already greater than 99%, meeting the selectivity requirements, the goal of directed evolution was to improve the reactivity of the mutants while maintaining high selectivity. Specifically, the EE value of the mutant with the highest relative activity in each round was measured. If the EE value was >99%, it was designated as the best mutant from that round of screening, and this was used to select the best mutant in each round.

[0064] Table 5 Results of the first round of directed evolution for model substrate S3

[0065] Enzyme mutants Relative activity ee(%) IR-117 1.00 >99 IR-117-S237T 1.09 Untested IR-117-S237G 0.37 Untested IR-117-S237C 1.23 Untested IR-117-S237V 1.02 Untested IR-117-A208Y 1.01 Untested IR-117-A208M 0.73 Untested IR-117-A208F 1.27 Untested IR-117-A208V 1.28 Untested IR-117-C192W 0.67 Untested IR-117-C192A 1.27 Untested IR-117-C192G 1.77 >99

[0066] (2) Using the IR-117-C192G mutant as the parent, site-saturation mutagenesis was performed on S67, H248, L216, V212, S244, N168, L252, and N65. Mutation library construction, protein expression, reaction screening, and detection were performed as described previously. The results of the second round of directed evolution are shown in Table 6.

[0067] Table 6 Results of the second round of directed evolution for model substrate S3

[0068]

[0069]

[0070] (3) Using the IR-117-C192G-N65S mutant as the parent, site-saturation mutagenesis was performed on S237, V174, H248, P124, Y243, and Q73. Mutation library construction, protein expression, reaction screening, and detection were performed as described previously. The results of the third round of directed evolution are shown in Table 7.

[0071] Table 7 Results of the third round of directed evolution for model substrate S3

[0072]

[0073]

[0074] (4) Using the IR-117-C192G-N65S-Q73A mutant as the parent, site-saturation mutagenesis was performed on E92, L216, G146, L75, and S244. Mutation library construction, protein expression, reaction screening, and detection were performed as described previously. The results of the fourth round of directed evolution are shown in Table 8.

[0075] Table 8 Results of the fourth round of directed evolution for model substrate S3

[0076] Enzyme mutants Relative activity ee(%) IR-117-C192G-N65S-Q73A 1.00 >99 IR-117-C192G-N65S-Q73A-E92E 0.99 Untested IR-117-C192G-N65S-Q73A-E92Q 1.04 Untested IR-117-C192G-N65S-Q73A-L216L 1.02 Untested IR-117-C192G-N65S-Q73A-G146T 1.05 Untested IR-117-C192G-N65S-Q73A-G146Y 1.01 Untested IR-117-C192G-N65S-Q73A-L75I 0.99 Untested IR-117-C192G-N65S-Q73A-S244T 1.07 Untested IR-117-C192G-N65S-Q73A-L216M 1.17 >99

[0077] (5) Using the IR-117-C192G-N65S-Q73A-L216M mutant as the parent, site-saturation mutagenesis was performed on D99 and L170. Mutation library construction, protein expression, reaction screening, and detection were performed as described previously. The results of the fifth round of directed evolution are shown in Table 9.

[0078] Table 9 Results of the fifth round of directed evolution for model substrate S3

[0079] Enzyme mutants Relative activity ee(%) IR-117-C192G-N65S-Q73A-L216M 1.00 >99 IR-117-C192G-N65S-Q73A-L216M-D99V 0.81 Untested IR-117-C192G-N65S-Q73A-L216M-D99L 0.86 Untested IR-117-C192G-N65S-Q73A-L216M-L170C 1.21 Untested IR-117-C192G-N65S-Q73A-L216M-L170V 0.93 Untested IR-117-C192G-N65S-Q73A-L216M-L170F 1.30 >99

[0080] Five rounds of directed evolution of IR-117 using the model substrate S3 resulted in the optimal mutant, IR-117-C192G-N65S-Q73A-L216M-L170F (hereinafter referred to as V5), which was derived from wild-type IR-117 with cysteine ​​at position 192 mutated to glycine, asparagine at position 65 mutated to serine, glutamine at position 73 mutated to alanine, leucine at position 216 mutated to methionine, and leucine at position 170 mutated to phenylalanine. The amino acid sequence is shown in SEQ ID No. 2. Compared to wild-type IR-117, V5 increased the yield of the model reaction from 6% to 15%, maintaining an EE value greater than 99%.

[0081] Example 3: Optimization of reaction conditions for mutant V5

[0082] 1. Preparation of Purified V5 Enzyme

[0083] Transform the V5 plasmid into TSR2566 competent cells and culture them in an inverted manner on LB solid medium containing kanamycin at 37°C for 12-16 hours. Pick a single colony and inoculate it into 4 mL LB-Kan liquid medium and activate it at 37°C, 250 rpm overnight. Transfer the activated bacterial solution to TB-Kan liquid medium at a 2% inoculum volume and culture it at 37°C, 220 rpm on a shaker for 3 hours until the OD reaches 0. 600 When the protein reaches 0.6-0.8, add IPTG at a final concentration of 0.5mM and induce expression at 22°C, 150rpm for 18h. After expression is completed, enrich the bacteria by low-temperature centrifugation. Resuspend with potassium phosphate buffer (pH 7.0, 100mM). Add protease inhibitor PMSF and lysozyme (McLean) at a final concentration of 1mg / mL in sequence and incubate at 37°C for 30min. After incubation, the protein is crushed by high-pressure homogenizer for 30min to obtain crude enzyme solution. Centrifuge the crude enzyme solution at 4°C, 12000g for 60min, and purify the supernatant after filtering through a 0.22μm cellulose acetate filter membrane.

[0084] Protein purification was performed using nickel affinity chromatography and The protein purification system was used. First, the nickel column was equilibrated with a loading and equilibration buffer (20mM potassium phosphate buffer, 25mM imidazole, pH 7.0), and then the crude enzyme solution was loaded at a loading flow rate of 0.5mL / min. After equilibration again, gradient elution was performed with elution buffer (20mM potassium phosphate buffer, 250mM imidazole, pH=7.0) in different proportions. The eluate containing the target protein was collected and concentrated by ultrafiltration using an ultrafiltration tube (10kDa molecular weight cutoff, AmiconUltra, Sigma Millipore). The residual imidazole was replaced with 20mM potassium phosphate buffer (pH 7.0) to obtain a pure enzyme solution. The protein concentration was determined using a modified BCA protein concentration assay kit (Shanghai Shenggong). The purified protein was divided into 50μL per tube and frozen with liquid nitrogen and stored at -80℃ for later use.

[0085] Protein expression results of pure enzyme: see Figure 4 .

[0086] 2. Optimization of reaction conditions for mutant V5

[0087] To further improve the reaction activity, using S3 as a model substrate, a systematic investigation was conducted focusing on the type and ratio of cosolvent, buffer type and pH, reaction temperature, reaction time, substrate concentration, surfactant type, and catalyst form. Relative activity, analytical yield, and EE values ​​were measured, where relative activity = product concentration under optimized conditions / product concentration under initial conditions. The results of the optimization study are shown in Table 10. Based on these results, the optimal reaction conditions are determined in Table 11.

[0088] Table 10 Results of optimizing reaction conditions of the dominant mutants for model substrate S3

[0089]

[0090]

[0091] Table 11 Optimal reaction conditions for the dominant mutants to catalyze the synthesis of substrate S3 into product P3

[0092]

[0093] Under optimal reaction conditions, the modified IR-117 mutant V5 increased the analytical yield of product P3 from 15% to 59%, while maintaining an ee value greater than 99%. The analytical yields before and after reaction optimization are shown in Table 12.

[0094] Table 12 Results of the investigation before and after optimization of reaction conditions for the dominant mutant of model substrate S3

[0095]

[0096] Chiral separation conditions and detection original spectrum:

[0097] The chiral separation structure of P3 is as follows Figure 5 The chiral detection column used was a Daicel chiralpak IG (4.6×250 mm, 5 μm), and the mobile phase ratio was acetonitrile / water = 30:70, 0.8 mL / min.

[0098] Example 4: Investigation of substrate applicability of mutant V5

[0099] The optimal reaction conditions described in Example 3 were adopted, and the reaction substrate S3 was replaced by 4-fluoro-α-phenyleneamide (S10), 3-fluoro-α-phenyleneamide (S14), 4-methyl-α-phenyleneamide (S16), 3-methyl-α-phenyleneamide (S26), 4-bromo-α-phenyleneamide (S13), N-(3H-indenyl)acetamide (S23), N-(5-fluoro-3H-indenyl)acetamide (S28), N-(3,4-dihydronaphthalene-1 -yl)acetamide (S9), N-[1-(naphthalen-1-yl)vinyl]acetamide (S27), N-[1-(thiophen-2-yl)vinyl]acetamide (S24), N-[1-(thiophen-3-yl)vinyl]acetamide (S29) and N-[1-phenylprop-1-en-2-yl]acetamide (S7). The catalytic activity of the dominant mutant V5 was investigated, and the corresponding asymmetric hydrogenation products were obtained in good yields or enantioselectivities. The detailed screening results are shown in Table 13.

[0100] Table 13 Results of investigation on substrate extension of mutant V5

[0101]

[0102]

[0103] Note: Some conditions have been adjusted according to different reactions. Please see the note column in Table 13 for details.

Claims

1. An imine reductase mutant, characterized in that: It is derived from Sinorhizobium Sinorhizobium The imine reductase IR-117 of the parent is used as a parent, and a mutant is obtained by mutation, wherein the cysteine ​​at position 192 of the parent is mutated to glycine, resulting in the mutant IR-117-C192G; The amino acid sequence of imine reductase IR-117 is shown in SEQ ID No.

1.

2. An imine reductase mutant, characterized in that: The mutant is a mutant obtained by taking the mutant IR-117-C192G described in claim 1 as a parent and causing mutation. The mutant is a mutant IR-117-C192G-N65S obtained by mutating the asparagine at position 65 of the parent to serine.

3. An imine reductase mutant, characterized in that: The mutant is a mutant obtained by taking the mutant IR-117-C192G-N65S described in claim 2 as a parent and causing a mutation. The mutant is obtained by mutating the 73rd glutamine of the parent to alanine to obtain the mutant IR-117-C192G-N65S-Q73A.

4. An imine reductase mutant, characterized in that: The mutant is a mutant obtained by taking the mutant IR-117-C192G-N65S-Q73A described in claim 3 as a parent and causing a mutation. The mutant is obtained by mutating the leucine at position 216 of the parent to methionine to obtain the mutant IR-117-C192G-N65S-Q73A-L216M.

5. An imine reductase mutant, characterized in that: The mutant is a mutant obtained by using the mutant IR-117-C192G-N65S-Q73A-L216M described in claim 4 as a parent, wherein the leucine at position 170 of the parent is mutated to phenylalanine to obtain the mutant IR-117-C192G-N65S-Q73A-L216M-L170F, and its amino acid sequence is shown in SEQ ID No.

2.

6. A DNA molecule, characterized in that: The DNA molecule encodes the imine reductase mutant according to any one of claims 1 to 5.

7. An expression vector, characterized in that: The expression vector contains the DNA molecule according to claim 6.

8. A host cell, characterized in that: The host cell contains the expression vector according to claim 7.

9. Use of the imine reductase mutant according to any one of claims 1 to 5, the DNA molecule according to claim 6, the expression vector according to claim 7, or the host cell according to claim 8 in catalytic asymmetric reduction to prepare α-arylacetamides and their derivatives; The α-arylacetamide and its derivatives are shown below: 、 、 、 、 、 、 。 10. The use according to claim 9, characterized in that: The preparation conditions are: temperature: 30° C., time: 72 h, and pH value: 6.0.

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