Imine reductase or mutant thereof and application of imine reductase or mutant thereof in preparation of alpha-arylacetamide and derivatives thereof through catalytic asymmetric reduction

Abstract

The invention provides an imine reductase mutant, which is a mutant obtained by taking imine reductase IR-117 from Sinorhizobium as a parent and carrying out mutation on the imine reductase IR-117. The mutant has the following sequence or an amino acid sequence which has at least 99% of identity in the following sequence and has imine reductase activity. Cysteine at the 192 position of a parent is mutated into glycine, and a mutant IR-117-C192G is obtained; the amino acid sequence of the imine reductase IR-117 is as shown in SEQ ID No. 1. The invention also provides application of the imine reductase IR-117 and the imine reductase mutant in preparation of alpha-arylacetamide and derivatives thereof through catalytic asymmetric reduction. According to the present invention, the imine reductase active center is adopted to promote the enamine-imine tautomerization of the substrate, such that the asymmetric hydrogenation reaction of the enamide can be highly selectively achieved under the aerobic condition in cooperation with the NADPH-mediated hydrogen transfer process, and the economic and green application scheme is provided for obtaining the high-value chiral aliphatic amine.

Description

Technical Field

[0001] The invention relates to an imine reductase or a mutant thereof and application 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 used 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 olefins, 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 chiral amines with multiple stereocenters because of its high efficiency, ease of operation, simple post-processing and easy industrial scale-up (Synthesis, 2021, 53, 193-214).

[0003] At present, the asymmetric hydrogenation of enamides catalyzed by transition metals such as rhodium, rubidium, and iridium is the main means to achieve asymmetric reduction of various enamide substrates. Since the first case of asymmetric hydrogenation of α-aryl enamides catalyzed by Rh(Ⅰ)-DIOP complex was reported by Kagan and Dang in 1972 (J.Am.Chem.Soc, 2002, 94, 6429-6433), the research on this reaction has been widely used in the construction of macromolecular skeletons of many types of drugs. Zhang Xumu's research group reported a new type of DIOP ligand that can be used for rhodium-catalyzed asymmetric hydrogenation of enamides, which has extremely high reactivity and a wide substrate spectrum (J.Org.Chem, 2000, 65, 5871). However, in this process, a high rhodium catalyst loading (2% mol) is required and excellent stereoselectivity can only be maintained under 10atm H2. Subsequently, Zhou Qilin et al. further developed a new monodentate phosphinamide ligand SiPhos, which achieved efficient asymmetric reduction of α-aryl enamides and their derivatives, with ee values ​​and yields greater than 99% (Angew. Chem. Int. Ed, 2002, 41, 2348). However, the reaction conditions are harsh, and toluene is required as the reaction solvent, and the hydrogen loading is increased to 50 atm at 5 ° C to maintain a high yield and selectivity. In summary, the transition metal-catalyzed enamide asymmetric hydrogenation method generally has harsh reaction conditions, requires the participation of noble metals and complex chiral ligands, uses organic solvents with high environmental costs, and metal residue problems and other factors that are not conducive to large-scale industrial production.

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

[0005] The invention provides an imine reductase or a mutant thereof and application of the imine reductase in preparing alpha-arylacetamide and derivatives thereof by catalytic asymmetric reduction.

[0006] Imine reductase is a NADPH-dependent oxidoreductase that can catalyze the asymmetric hydrogenation of the potential chiral C=N bond of imines, especially cyclic imines, to form corresponding chiral amine compounds. The present invention is based on the enzymatic enamine-imine tautomerism and hydrogen transfer mechanism, with α-phenylenamide as the substrate and imine reductase as the biocatalyst, as the reaction path for asymmetric reduction to prepare α-arylacetamide and its derivatives; construct a candidate enzyme library, screen the imine reductase using a model reaction, and obtain an enzyme with initial reaction activity; construct a mutant library of different amino acid residue sites, and use directed evolution to screen the reaction activity and selectivity of the mutants; obtain an advantageous mutant with high reactivity and high selectivity, and investigate the applicable range of its reaction substrate.

[0007] The present invention provides an imine reductase mutant, which is a mutant obtained by taking imine reductase IR-117 from Sinorhizobium as a parent and undergoing mutation, 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:

[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 with IR-117-C192G as a parent, and 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:

[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] Further preferably, it is a mutant obtained by mutation of IR-117-C192G-N65S-Q73A-L216M as a parent, and 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:

[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, wherein the DNA molecule encodes the imine reductase mutant.

[0019] The invention provides an expression vector, which contains the DNA molecule.

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

[0021] The present invention also provides the 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] Further, 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, wherein the α-arylacetamide and its derivatives are synthesized by using 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. 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] Further, 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, in the reaction conditions, 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 NADPH-mediated hydrogen transfer process under aerobic conditions to achieve asymmetric hydrogenation reaction of enamide 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 model substrate S3 corresponding to the product P3 detected by LC-MS;

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

[0038] Figure 5 The chiral detection original spectrum of the product P3 generated by the mutant V5 catalyzing the S3 reaction 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 mutant V5 catalyzing the S3 reaction). 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-177 was codon optimized, fully synthesized, and constructed using E. coli as a host cell to obtain the wild-type IR-117 recombinant expression vector pET28a-IR-117 (vector map as shown in the figure). Figure 1 The expression vector was transformed into TSR2566 competent cells and cultured in an inverted manner on LB solid medium containing kanamycin at 37°C for 12-16h.

[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 LB-Kan liquid medium and activated overnight at 37°C and 250 rpm in a shaking incubator. 1 mL of the activated bacterial solution was inoculated into 50 mL TB-Kan liquid medium and incubated at 37°C and 220 rpm for 3 h. 600When the concentration reaches 0.6-0.8, place in a 4℃ refrigerator for 30 minutes, then add IPTG with a final concentration of 0.5mM, and induce expression for 18 hours at 22℃ and 150rpm. 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 and mix well, and then 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, full gene synthesis and vector construction were performed using Escherichia coli as the host cell to obtain a 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 inverted cultured at 37°C for 12-16h to construct a GDH expression strain. The protein expression method was the same as in Example 1-2 (the medium antibiotic was Amp). 600 = 30, and crushed by high pressure homogenizer at 4°C and 800 bar for 30 min to prepare crude GDH enzyme solution.

[0046] 4. Investigation of reaction activity

[0047] The wild-type imine reductase IR-117 constructed and expressed in the early stage was used to investigate the initial reaction activity of the model substrate α-phenylene amide (hereinafter referred to as S3). The specific operation is as follows: 200 μL IRED crude enzyme solution, 200 μL GDH crude enzyme solution, 80 μL NADP + Sodium salt (1.875mM, Kpi solution) and D-Glucose (187.5mM, KPi solution) mixed solution and 40μL substrate S3 (15mM, DMSO solution) were added to a 2mL reaction bottle and shaken at 800rpm at 25°C 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 analysis 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 calculated to make a standard curve (hereinafter referred to as the analysis yield). The standard curve is attached. Figure 3The chirality calculation is expressed as ee value, ee = ((S, SR, R) / (S, S + R, R)) * 100%. The screening results of the initial activity 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 parent and model substrate S3 1. Construction of IR-117 mutant library

[0051] Using IR-117 as the parent, S237, A208, and C192 were subjected to point saturation mutation. First, the pET28a-IR-117 plasmid was used as a template, and the corresponding degenerate primers were designed by the 22codon trick method for PCR reaction. The PCR reaction used 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 excision. The nucleic acid concentration of the gel recovered products was determined by ultra-micro nucleic acid analyzer and the DNA seamless cloning kit ( IIOne Step Cloning Kit, Novozyme) 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 min, take the incubation product and transform it into TSR2566 competent cells, and place it in an incubator at 37°C for 12-16 hours. Pick a single colony on the plate and inoculate it into a 96-well plate containing 300 μL LB-Kan liquid medium. Activate it in a shaker at 37°C and 250 rpm overnight. Pipette 50 μL of the activated bacterial solution and transfer it to a 96-deep-well plate containing 900 μL TB-Kan liquid medium, and shake it for 3 hours at 37°C and 250 rpm. Then, cool the 96-well plate in an ice bath for 30 min, add 50 μL IPTG stock solution (10 mM) to induce expression, and culture it at 22°C and 200 rpm for 18 hours. Centrifuge the cells at low temperature, add 100 μL potassium phosphate buffer (pH 7.0, 100 mM) by pipetting, and vortex to resuspend the cells. Then add 100 μL of the prepared GDH crude enzyme solution and 40 μL of NADP + A mixed solution of sodium salt (1.875 mM, KPi solution) and D-Glucose (187.5 mM, KPi solution) and 20 μL of substrate S3 (15 mM, DMSO solution) were sealed and reacted at 25°C and 800 rpm for 24 h. After the reaction, 900 μL of anhydrous ethanol was added and the reaction activity was determined by LC-MS.

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

[0063] Since the EE value of IR-117 in the initial screening results was greater than 99%, meeting the selectivity requirements, the goal of directed evolution was to improve the reaction activity of the mutants while maintaining high selectivity. That is, the EE value was measured for the mutants that met the highest relative activity in each round. If the EE value was >99%, it could be used as the best mutant obtained in that round of screening, and so on, as the basis for selecting the best mutants 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, point saturation mutations were performed on S67, H248, L216, V212, S244, N168, L252, and N65. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. 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, point saturation mutations were performed on S237, V174, H248, P124, Y243, and Q73. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. 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, point saturation mutations were performed on E92, L216, G146, L75, and S244. The mutant library construction, protein expression, reaction screening, and detection methods were the same as described above. 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, D99 and L170 were subjected to point saturation mutation. The mutant library construction, protein expression, reaction screening and detection methods were the same as described above. 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] Under the condition of model substrate S3, IR-117 was subjected to 5 rounds of directed evolution, and the optimal mutant IR-117-C192G-N65S-Q73A-L216M-L170F (hereinafter collectively referred to as V5) was screened: the wild-type IR-117 was used as the parent, and the cysteine ​​at position 192 was mutated to glycine, the asparagine at position 65 was mutated to serine, the glutamine at position 73 was mutated to alanine, the leucine at position 216 was mutated to methionine, and the leucine at position 170 was mutated to phenylalanine. The amino acid sequence is shown in SEQ ID No. 2. Compared with the wild-type 1R-117, V5 can promote the model reaction yield from 6% to 15%, and the ee value is maintained at more 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 incubate them on LB solid medium containing kanamycin at 37°C for 12-16 hours. Pick a single clone 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 and incubate it at 37°C, 220 rpm for 3 hours until the OD reaches 0. 600 When it reaches 0.6-0.8, add IPTG with a final concentration of 0.5mM, and induce expression at 22°C, 150rpm for 18h. After the expression is completed, centrifuge at low temperature to enrich the bacteria. Resuspend with potassium phosphate buffer (pH 7.0, 100mM). Add protease inhibitor PMSF and lysozyme (McLean) with a final concentration of 1mg / mL in sequence, and incubate at 37°C for 30min. After the incubation is completed, 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 by nickel affinity chromatography and The protein purification system was used. First, the nickel column was balanced with a loading and equilibration buffer (20mM potassium phosphate buffer, 25mM imidazole, pH 7.0), and then the crude enzyme solution was loaded with 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, ultrafiltered and concentrated with an ultrafiltration tube (10kDa molecular weight cutoff, AmiconUltra, Sigma Millipore), and 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°C for use.

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

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

[0087] In order to further improve the reaction activity, S3 was used as a model substrate to systematically investigate the type and proportion of cosolvents, buffer type and pH, reaction temperature, reaction time, substrate concentration, surfactant type and catalyst form. The relative activity, analytical yield and ee value of the reaction were detected, where relative activity = product concentration under optimized conditions / product concentration under initial conditions. The results of the investigation of the optimization of reaction conditions are shown in Table 10. According to the investigation 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 synthesis of product P3 by substrate S3 catalyzed by the dominant mutant

[0092]

[0093] Under the optimal reaction conditions, the modified IR-117 mutant V5 can increase the analytical yield of product P3 from 15% to 59%, and the ee value is still maintained at more than 99%. The analytical yields before and after the reaction optimization are shown in Table 12.

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

[0095]

[0096] Chiral separation conditions and detection original spectra:

[0097] The chiral separation structure of P3 is as follows Figure 5 The chiral detection column used was 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 suitability 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 with 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] Remarks: Some conditions have been adjusted according to different reactions, see the Remarks column of Table 13 for details.

[0104] 4. Sequence Listing (Amino Acids)

[0105] SEQ ID No.1 (pIR117):

[0106] MQPAISVLGTGRMGSALAYALLKAGHPTTVWNRTPAKAAPLAAAGAEVA

[0107] ASVRNAVAVSEVVIVNVSDYQATQSLLRDKEVAGALEGRLIIELTSGTPDG

[0108] GREAHGWAQRQGARYLDGAILATPDFIGTEAGTLLVSGPSGVFEEGRNVL

[0109] GALGGNVQFIGEDPGLANALDSAVLALMWGALFGALQSIAVCRAEAIDL

[0110] GVLARQWTATAPVVEGLVSDLIKRSAAGRYDADAETLSSVSPHYSAFHHL

[0111] VDLMEARGIDRTITGGYEAIFRRAIEAGHLHDDFASLSQFMGQPASEQ ID No.2(IR117-C192G-N65S-Q73A-L216M-L170F):MQPAISVLGTGRMGSALAYALLKAGHPTTVWNRTPAKAAPLAAAGAEVAASVRNAVAVSEVVIVSVSDYQATASLLRDKEVAGALEGRLIIELTSGTPDGGREAHGWAQRQGARYLDGAILATPDFIGTEAGTLLVSGPSGVFEEGRNVLGALGGNVQFIGEDPGLANAFDSAVLALMWGALFGALQSIAVGRAEAIDLGVLARQWTATAPVVEGMVSDLIKRSAAGRYDADAETLSSVSPHYSAFHHLVDLMEARGIDRTITGGYEAIFRRAIEAGHLHDDFASLSQFMGQPA。

Claims

1. An imine reductase mutant, characterized in that: The invention relates to a mutant obtained by taking the imine reductase IR-117 from Sinorhizobium as a parent and subjecting it to mutation. 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 was mutated to glycine to obtain the mutant IR-117-C192G; The amino acid sequence of imine reductase IR-117 is shown in SEQ ID No.

1.

2. The imine reductase mutant according to claim 1, characterized in that: It is a mutant obtained by mutation with IR-117-C192G as a parent, and 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 asparagine at position 65 of the parent was mutated to serine to obtain the mutant IR-117-C192G-N65S.

3. The imine reductase mutant according to claim 2, characterized in that: It is a mutant obtained by mutation of IR-117-C192G-N65S as a parent, and 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 glutamine at position 73 of the parent was mutated to alanine to obtain the mutant IR-117-C192G-N65S-Q73A.

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

5. The imine reductase mutant according to claim 4, characterized in that: It is a mutant obtained by mutation of IR-117-C192G-N65S-Q73A-L216M as a parent, and 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 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.

6. A DNA molecule, characterized in that: The DNA molecule encodes the imine reductase mutant according to any one of claims 1 to 4.

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 imine reductase IR-117, 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 catalyzing asymmetric reduction to prepare α-arylacetamide and its derivatives; The α-aryl acetamide and its derivatives are shown in Formula I and Formula IV: in, 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; Further, the C1-C6 substituted phenyl group is selected from a halogen atom and a methyl group; Furthermore, the heterocyclic substituent is selected from common heterocyclic structures of thiophene.

10. A method for preparing α-arylacetamide and its derivatives by catalytic asymmetric reduction, characterized in that: It uses formula I as a substrate, imine reductase IR-117, the imine reductase mutant described in any one of claims 1 to 5, the DNA molecule described in claim 6, the expression vector described in claim 7, or the host cell described in claim 8 as a catalyst to synthesize α-arylacetamide and its derivatives. The synthesis route is as follows: 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; Further, the C1-C6 substituted phenyl group is selected from a halogen atom and a methyl group; Further, the heterocyclic substituent is selected from common heterocyclic structures of thiophene; Preferably, in the reaction conditions, the reaction temperature is 30° C., the reaction time is 72 h, and the pH value is 6.0.

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