Imine reductase or its mutant and use thereof in synthesizing polycyclic chiral nitrogen heterocyclic compounds, method for synthesizing polycyclic chiral nitrogen heterocyclic compounds

Abstract

The present invention relates to an imine reductase or a mutant thereof and an application thereof in synthesizing a polycyclic chiral nitrogen heterocyclic compound, a method for synthesizing a polycyclic chiral nitrogen heterocyclic compound, and belongs to the field of enzyme engineering. The present invention provides an imine reductase IR-F7 mutant, which is a mutant obtained by mutation of a wild-type imine reductase IR-F7 with an amino acid sequence such as shown in SEQ ID No.1 as a parent. The present invention provides an application of an imine reductase IR-F7 or a mutant thereof, an imine reductase IR-B8 or a mutant thereof in synthesizing a polycyclic chiral nitrogen heterocyclic compound. The imine reductase or a mutant thereof of the present invention can catalyze intramolecular reductive amination, synthesize a polycyclic chiral nitrogen heterocyclic in one step, and avoid the use of expensive chiral reagents; by combining the DKR process, the raw materials can be fully utilized, no additional amino donor is required, and a good yield and selectivity are obtained; it has a wider substrate application range, and has more potential in the industrial application of synthesizing polycyclic chiral nitrogen heterocyclic compounds.

Description

Technical Field

[0001] The invention relates to imine reductase or a mutant thereof and application thereof in synthesizing polycyclic chiral nitrogen heterocyclic compounds, and a method for synthesizing polycyclic chiral nitrogen heterocyclic compounds, belonging to the field of enzyme engineering. Background Art

[0002] Polycyclic chiral nitrogen heterocycles refer to organic compounds composed of two or more cyclic structures (including carbon rings and nitrogen heterocycles) and containing at least one chiral center. This type of structure is easy to modify and widely exists in a variety of biologically active molecules. It is a key intermediate in the synthesis of a variety of alkaloids and chiral drugs. At present, the synthesis of polycyclic chiral nitrogen heterocyclic compounds mainly includes two chemical methods: chiral resolution and asymmetric synthesis. The stereoselective control is the technical key in the synthesis process.

[0003] Chiral separation of racemates is one of the most widely used methods in the synthesis of chiral compounds. This strategy usually uses appropriate aliphatic or aromatic compounds as raw materials, forms a ring structure through a series of chemical reactions, and finally obtains the chiral product through special chiral separation reagents. Yan et al. ( Synth. Commun. 2017, 47 , 238-244) This method was used to synthesize azabicyclic compounds, using 2,3-pyridinedicarboxylic acid and its derivatives as starting materials, through two-step reactions of intramolecular cyclization and catalytic hydrogenation reduction, and multiple chemical splitting under the condition of adding tartaric acid, finally obtaining chiral azabicyclic compounds. However, through chiral splitting, at most only half of the target configuration product can be obtained, and the other half of the isomers are wasted because they cannot be converted, resulting in poor raw material utilization. In addition, the special chiral splitting reagents and equipment are expensive, the separation process is time-consuming, and the yield and purity are difficult to reach the ideal level.

[0004] Asymmetric synthesis is the process of introducing chiral centers during the synthesis process by using chiral catalysts, which mainly include metal catalysis and organic catalysis. For example, Wang et al. used chiral palladium catalysts to catalyze the asymmetric addition reaction of olefins and amine compounds to generate chiral nitrogen-containing heterocyclic compounds ( Org. Process Res. Dev. 2022, 26 ,2510-2518). However, this synthesis strategy requires the use of expensive metal catalysts and relatively harsh reaction conditions, which is costly and not very applicable. Organic catalysis uses chiral organic small molecules (such as chiral phosphines) as catalysts to achieve chiral induction and selective control through interactions such as hydrogen bonds. This method avoids the use of metal catalysts and reduces environmental pollution. However, there are currently few types of chiral catalysts used in the synthesis of nitrogen-containing mid-ring compounds, and they are expensive and have low substrate universality ( Pure Appl. Chem . 2014, 86 , 1217-1226).

[0005] At present, the methods for biocatalytic synthesis of multi-ring chiral nitrogen heterocyclic compounds are very limited. Application number: 201410418987.9, invention name: A 3-aminopyrrolidine compound and its synthesis method and use. Lin et al. reported using transaminase as a catalyst, under the action of an amino donor, to obtain an amino-substituted product, and then undergoing an asymmetric reductive amination reaction to obtain a chiral nitrogen heterocyclic compound. However, since the reaction catalyzed by transaminase is reversible, an excess of amino donors is usually required. Moreover, the substrate range of this synthesis strategy is narrow, and it can only catalyze the synthesis of some five- and six-membered nitrogen heterocyclic compounds. Patents ZL202311528667.4 and 202311439586.7 also disclose the following technical scheme: Using imine reductase to synthesize the chiral side chain of moxifloxacin ( S , S )-2,8-diazabicyclo[4,3,0]nonane, but its catalytic synthesis has limited substrate structures and low universality, which restricts its application in a wider range of substrates. Summary of the invention

[0006] The present invention provides an imine reductase and a mutant thereof to solve the industrial application problem in synthesizing polycyclic chiral nitrogen heterocyclic compounds. The present invention also provides the use of the imine reductase and the mutant thereof.

[0007] The present invention provides an imine reductase mutant, which is a mutant obtained by mutation of the wild-type imine reductase IR-F7 with an amino acid sequence as shown in SEQ ID No. 1 as a parent, wherein the mutant is one of the following or an amino acid sequence having at least 90% identity with any sequence and having imine reductase activity:

[0008] The threonine at position 73 of the parent was mutated to aspartic acid to obtain the mutant F7-T73D;

[0009] The threonine at position 73 of the parent was mutated to alanine to obtain the mutant F7-T73A;

[0010] The methionine at position 125 of the parent was mutated to arginine to obtain the mutant F7-M125R;

[0011] The methionine at position 19 of the parent was mutated to leucine to obtain the mutant F7-M19L;

[0012] The threonine at position 99 of the parent was mutated to alanine to obtain the mutant F7-T99A;

[0013] The leucine at position 97 of the parent was mutated to arginine to obtain the mutant F7-L97R;

[0014] The alanine at position 123 of the parent was mutated to leucine to obtain the mutant F7-A123L;

[0015] The isoleucine at position 124 of the parent was mutated to valine to obtain the mutant F7-I124V;

[0016] The isoleucine at position 124 of the parent was mutated to leucine to obtain the mutant F7-I124L;

[0017] The proline at position 128 of the parent was mutated to threonine to obtain the mutant F7-P128T;

[0018] The alanine at position 239 of the parent was mutated to arginine to obtain the mutant F7-A239R.

[0019] Furthermore, it is a mutant obtained by mutation with F7-I124V as a parent, wherein the mutant is the following sequence or an amino acid sequence having at least 90% identity with the following sequence and having imine reductase activity:

[0020] The proline at position 128 of the parent was mutated to cysteine ​​to obtain the mutant F7-I124V-P128C.

[0021] Furthermore, it is a mutant obtained by mutation of F7-I124V-P128C as a parent, wherein the mutant is one of the following or an amino acid sequence having at least 90% identity with any sequence and having imine reductase activity:

[0022] The methionine at position 183 of the parent was mutated to phenylalanine to obtain the mutant F7-I124V-P128C-M183F;

[0023] The tyrosine at position 221 of the parent was mutated to isoleucine to obtain the mutant F7-I124V-P128C-Y221I;

[0024] The leucine at position 187 of the parent was mutated to phenylalanine to obtain the mutant F7-I124V-P128C-L187F;

[0025] The threonine at position 240 of the parent was mutated to isoleucine to obtain the mutant F7-I124V-P128C-T240I;

[0026] The threonine at position 240 of the parent was mutated to leucine to obtain the mutant F7-I124V-P128C-T240L;

[0027] Among them, the amino acid sequence of the mutant F7-I124V-P128C-T240L is shown in SEQ ID NO.4.

[0028] The present invention provides a DNA molecule, wherein the DNA molecule encodes the imine reductase mutant.

[0029] The invention provides an expression vector, which contains the DNA molecule.

[0030] The present invention provides a host cell, wherein the host cell contains the expression vector.

[0031] The present invention provides use of imine reductase IR-F7, the imine reductase mutant, imine reductase IR-B8 or its mutant, the DNA molecule, the expression vector, or the host cell in synthesizing polycyclic chiral nitrogen heterocyclic compounds; the amino acid sequence of the imine reductase IR-B8 is shown in SEQ ID No. 2, and the structural formula of the polycyclic chiral nitrogen heterocyclic compound is shown in Formula I or Formula II:

[0032]

[0033] Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; X=CH2, NBn, NBz, NBoc;

[0034]

[0035] Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; R1=F, Cl, Br, I, CH3, CH3O.

[0036] The present invention provides a method for synthesizing a polycyclic chiral nitrogen heterocyclic compound, which uses formula III or formula IV as a substrate, and uses imine reductase IR-F7 or the imine reductase mutant, imine reductase IR-B8 or its mutant as a biocatalyst to synthesize the polycyclic chiral nitrogen heterocyclic compound. The synthesis route is shown in general formula I or general formula II:

[0037]

[0038] Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; X=CH2, NBn, NBz, NBoc;

[0039]

[0040] Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; R1=F, Cl, Br, I, CH3, CH3O.

[0041] The reaction system contains coenzymes, glucose and glucose dehydrogenase.

[0042] Preferably, the coenzyme is NADP +Salt.

[0043] Existing methods for synthesizing polycyclic chiral nitrogen heterocyclic compounds include chemical methods and biocatalytic methods. The chemical method has disadvantages such as complex synthesis process, poor raw material utilization, high cost, and environmental pollution. Biocatalysis has good selectivity, milder reaction, and reduced environmental pressure. However, the current technical solutions do not have a wide range of applicability in their substrate spectrum.

[0044] Imine reductase (IRED) is a class of NAD(P)H-dependent oxidoreductases with high catalytic efficiency and strong regio- and stereo-selectivity. By utilizing the excellent selectivity of imine reductase and combining it with the dynamic kinetic resolution (DKR) of reaction intermediates, racemic substrates can be converted into products of target configuration as much as possible. Based on this, a general and efficient method for synthesizing polycyclic chiral nitrogen compounds was established by integrating the intramolecular reductive amination and DKR process catalyzed by imine reductase.

[0045] The beneficial effects of the present invention are:

[0046] The present invention provides an imine reductase and a mutant thereof, which can catalyze intramolecular reductive amination to synthesize polycyclic chiral nitrogen heterocycles in one step, thus avoiding the use of expensive chiral reagents; by combining with a DKR process, the raw materials can be fully utilized, no additional amino donor is required, and a good yield and selectivity can be obtained; meanwhile, the method has a wider substrate application range and has greater potential in the industrial application of synthesizing polycyclic chiral nitrogen heterocycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the recombinant plasmid map of wild-type IR-F7;

[0048] Figure 2 is the recombinant plasmid map of wild-type IR-B8;

[0049] Figure 3 This is the standard curve of the model substrate 7a corresponding to the product 7b detected by LC-MS;

[0050] Figure 4 The protein map of M3 mutant detected by SDS-PAGE;

[0051] Figure 5 This is the chiral detection spectrum of the product 7b generated by the reaction of 7a catalyzed by M3 under optimal conditions (where A is the original chiral detection spectrum of the racemate 7b; B is the original chiral detection spectrum of the product 7b generated by the reaction of 7a catalyzed by M3: 99% ee). DETAILED DESCRIPTION

[0052] Example 1 Construction of expression strain and investigation of initial reaction activity

[0053] 1. Construction of wild-type imine reductase expression strain

[0054] For those coming from Streptomyces ipomoeae Wild-type imine reductase IR-F7, amino acid sequence as shown in SEQ ID No. 1, codon optimization was performed using Escherichia coli as the host, and a recombinant expression vector containing the IR-F7 coding gene was obtained by whole gene synthesis and vector construction, named pET28a-IR-F7. The gene map is referenced Figure 1 The pET28a-IR-F7 recombinant expression vector was transformed into Escherichia coli TSR2566 strain (Qingke Biotechnology) to construct the IR-F7 expression strain.

[0055] For those coming from Labilithrix luteola Wild-type imine reductase IR-B8, the amino acid sequence of which is shown in SEQ ID No. 2, was codon optimized using Escherichia coli as the host, and a recombinant expression vector containing the IR-B8 coding gene was obtained by full gene synthesis and vector construction, which was named pET28a-IR-B8. The gene map is referenced Figure 2 The pET28a-IR-B8 recombinant expression vector was transformed into Escherichia coli TSR2566 strain (Qingke Biotechnology) to construct the IR-B8 expression strain.

[0056] 2. Protein Expression of Imine Reductase and Preparation of Crude Enzyme Solution

[0057] The constructed IR-F7 and IR-B8 expression strains were inoculated into a shaking tube containing 3 mL of LB medium (LB-Kan) containing Kan resistance, and activated overnight in a shaker at 37°C and 200 rpm. Then, 1 mL of the activated bacterial solution was inoculated into a conical flask containing 50 mL of TB medium (TB-Kan) containing Kan resistance, and incubated at 37°C and 200 rpm for 2.5 h. 600 When the OD reached 0.6-0.8, the bacterial solution was placed on ice for 30 min and IPTG was added to a final concentration of 0.5 mM. Subsequently, the expression was induced at 22°C and 150 rpm for 20 h. After the induction expression was completed, the bacteria were collected by centrifugation and the supernatant was discarded. The bacterial pellet was resuspended in 100 mM potassium phosphate (KPi, pH 7.0) buffer and the OD was adjusted. 600 to 30 to obtain crude enzyme solution.

[0058] Construction of glucose dehydrogenase (GDH) expression strain, protein expression and crude enzyme solution preparation

[0059] Will come from Bacillus subtilisThe amino acid sequence of glucose dehydrogenase (GDH) shown in SEQ ID No. 3 was constructed into the prokaryotic expression vector pET-28a(+), and the restriction enzyme cleavage site inserted was N I and XOt I, transform TSR2566 E. coli competent cells to obtain GDH expression strain. The protein expression method is the same as that in Example 1-2. 600 = 30 whole cell resuspension, and then disrupted by high pressure homogenizer (4 ° C, 800 bar) for 30 min to obtain GDH crude enzyme solution.

[0060] 4. Investigation of initial reaction activity of imine reductase

[0061] Using previously expressed wild-type imine reductases IR-F7 and IR-B8, we investigated the biosynthesis of the model substrate 2-(4-aminobutyl)-3,4-dihydronaphthalen-1(2 H )-one (hereinafter collectively referred to as 7a) for initial reaction activity screening. The specific steps are as follows: 200 μL of treated whole cell enzyme solution (OD 600 =30), 200 μL of broken GDH enzyme solution (OD 600 =30), 80 μL NADP + Sodium salt (3.75 mM, KPi solution), 80 μL Glucose (375 mM, KPi solution), 40 μL substrate 7a (75 mM, EtOH solution), shake at 800 rpm for 24 h at room temperature. After the reaction, add 900 μL anhydrous ethanol, shake and mix, and then centrifuge. Take 400 μL of supernatant and add it to a 96-well ELISA plate for analysis and detection. The concentration of the product is determined by LC-MS detection of product standards with different concentration gradients and containing internal standards. The standard curve is calculated by statistically calculating the peak area ratio of the standard and the internal standard. The standard curve is shown in the figure. Figure 3 As shown. The analytical yield of the product = actual product concentration / theoretical product concentration (hereinafter referred to as analytical yield). Calculated by product peak area ee、dr value, ee The value is the ratio of the peak areas between products with different configurations of enantiomers. dr The value is the ratio of the peak areas of the two diastereoisomer products. ee =(( S , S - R , R ) / ( S , S + R , R ))*100%, dr= ( S , S ) / ( S , R ). The initial reactivity results are shown in Table 1.

[0062] Table 1 Initial reactivity against model substrate 7a

[0063] Enzyme name Microbial sources Accession numberGenebank Accession Analytical yield (%) （%） IR-F7 WP_009330409.1 8 70 >19:1 IR-B8 AKU97888.1 7 67 >19:1

[0064] Example 2 Directed evolution using wild-type IR-F7 as parent and model substrate 7a

[0065] 1. Construction of IR-F7 mutant library

[0066] Using IR-F7 as the parent, site-directed saturation mutagenesis was performed on amino acid residues such as L18, T73, I124V, M125, Y221, and D239. First, the pET28a-IR-F7 plasmid was used as a template, and the corresponding degenerate primers were designed using the 22 codon 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:

[0067] Table 2 PCR reaction system

[0068] Components Volume (50 μL) Plasmid template 1 μL KOD One TM PCR Master MIX-Blue (2×) 25 μL Upstream primer (10 μM) 2 μL Downstream primer (10 μM) 2 μL <![CDATA[ddH2O]]> 20 μL

[0069] PCR reaction conditions are shown in Table 3:

[0070] Table 3 PCR reaction conditions

[0071]

[0072] The linear DNA products obtained by PCR were subjected to 1% agarose gel electrophoresis and separated by gel excision and recovery. The nucleic acid concentration of the gel recovery products was determined by ultra-micro nucleic acid analyzer, and ligated according to the DNA seamless cloning kit (ClonExpress® II One Step Cloning Kit, Novozymes). The ligation system is shown in Table 4. The products were incubated at 37°C for 30 min, and the ligation products were transformed into TSR2566 Escherichia coli competent cells.

[0073] Table 4 PCR product ligation system

[0074] Components Volume (10 μL) Linear plasmid 2 μL Recombinase (Exnase Ⅱ) 1 μL CE Ⅱ Buffer (5×) 2 μL <![CDATA[ddH2O]]> 5 μL

[0075] 2. Reactivity screening

[0076] (1) Pick a single colony from the plate and inoculate it into a 96-well plate containing 300 µL LB-Kan liquid medium, and activate it overnight in a shaker at 37°C and 250 rpm. Take 50 μL of the activated bacterial solution and transfer it to a 96-deep-well plate containing 900 μL TB-Kan liquid medium, and culture it at 37°C and 250 rpm for 3 h. Place the 96-well plate on ice for 30 min, then add 50 μL IPTG stock solution (10 mM) to induce expression, and then culture it at 22°C and 150 rpm for 20 h. Collect the bacteria by low-temperature centrifugation, add 100 μL KPi (pH 7.0) buffer to each well, and vortex to completely resuspend the cells. Then add 100 μL of broken GDH enzyme solution (OD 600 =30), 40 μL NADP + Sodium salt (3.75 mM, KPi solution), 40 μL Glucose (375 mM, KPi solution), 20 μL substrate 7a (75 mM, EtOH solution), shake at 800 rpm for 24 h at room temperature. After the reaction, add anhydrous ethanol, shake and mix, and centrifuge. Take 400 μL of the supernatant for LC-MS detection.

[0077] The activity and ee The mutants with improved values ​​were cultured in shake flasks and their reaction activity was verified. The screening and detection methods for verifying the reaction activity were the same as those in Example 1-4. The relative activity of the reaction of substrate 7a, ee Value and dr 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.

[0078] Table 5 Results of the first round of directed evolution under the model substrate 7a

[0079] Enzymes Relative activity （%） dr F7 1.00 70 >19:1 F7-L18I 0.95 81 >19:1 F7-T73D 1.13 82 >19:1 F7-T73A 1.05 80 >19:1 F7-M125Q 1.21 65 >19:1 F7-M125R 1.04 87 >19:1 F7-M125N 0.94 91 3:1 F7-M183V 1.43 26 >19:1 F7-Y221T 1.30 54 >19:1 F7-Y221K 1.25 60 >19:1 F7-D238W 1.06 74 >19:1 F7-M19L 1.64 73 >19:1 F7-T99A 1.34 99 >19:1 F7-L97R 1.41 70 >19:1 F7-L97Y 1.31 72 5:1 F7-A123L 1.36 70 >19:1 F7-I124V 1.77 87 >19:1 F7-I124D 1.64 56 >19:1 F7-I124L 1.37 87 >19:1 F7-P128T 1.34 79 >19:1 F7-A239R 1.31 71 >19:1 F7-H244Q 1.46 65 >19:1

[0080] (2) Using the F7-I124V mutant as the parent, site-directed saturation mutagenesis was performed on the amino acid residues M19, P128, A208, and H250. 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 verification are shown in Table 6.

[0081] Table 6 Results of the second round of directed evolution under model substrate 7a

[0082] Enzyme mutants Relative activity （%） dr F7-I124V 1.00 87 >19:1 F7-I124V-A208E 0.90 43 >19:1 F7-I124V-A208V 1.06 23 >19:1 F7-I124V-H250D 1.02 19 >19:1 F7-I124V-H250W 0.88 17 >19:1 F7-I124V-P128G 0.92 43 >19:1 F7-I124V-P128C 0.99 90 >19:1 F7-I124V-M19G 0.97 6 >19:1

[0083] (3) Using the F7-I124V-P128C mutant as the parent, site-directed saturation mutagenesis was performed on H169, M183, Y221, T240, D246, H250, A208 and other sites. 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 verification are shown in Table 7.

[0084] Table 7 Results of the third round of directed evolution under the model substrate 7a

[0085] Enzyme mutants Relative activity （%） dr F7-I124V-P128C 1.00 90 >19:1 F7-I124V-P128C-M183F 1.18 90 >19:1 F7-I124V-P128C-M183D 1.18 30 8:1 F7-I124V-P128C-Y221R 1.16 12 >19:1 F7-I124V-P128C-Y221F 1.26 78 >19:1 F7-I124V-P128C-Y221S 1.17 33 >19:1 F7-I124V-P128C-Y221I 1.19 91 >19:1 F7-I124V-P128C-H169F 0.67 95 3:1 F7-I124V-P128C-L187P 1.10 89 3:1 F7-I124V-P128C-L187F 1.34 84 >19:1 F7-I124V-P128C-T240I 1.10 92 >19:1 F7-I124V-P128C-T240L 1.20 95 >19:1 F7-I124V-P128C-H250D 1.32 68 >19:1 F7-I124V-P128C-H250G 1.17 88 >19:1

[0086] Under the condition of model substrate 7a, IR-F7 was subjected to three rounds of directed evolution, and the optimal mutant F7-I124V-P128C-T240L (hereinafter collectively referred to as M3) was screened: the wild-type IR-F7 was used as the parent, the isoleucine at position 124 was mutated to valine, the proline at position 128 was mutated to cysteine, and the threonine at position 240 was mutated to leucine, and the amino acid sequence is shown in SEQ ID NO.4. Compared with the wild-type IR-F7, the yield of the catalytic model substrate 7a to synthesize product 7b was increased from 8% to 16%, ee Value increased from 70% to 95%, and dr The value is greater than 19:1.

[0087] Example 3 Optimization of reaction conditions for M3 mutant

[0088] 1. Preparation of M3 purified enzyme

[0089] The M3 plasmid was transformed into competent cells of E. coli TSR2566, spread on a plate containing Kan resistance, and inverted at 37°C for 12 h. A single clone colony was picked and inoculated into a conical flask containing 20 mL LB-Kan liquid medium, and activated overnight at 37°C and 200 rpm. The activated bacterial solution was transferred to TB-Kan liquid medium at a 2% inoculum and cultured at 37°C and 200 rpm for 3 h until the OD 600 The value reached 0.6-0.8. Add IPTG with a final concentration of 0.5 mM, and induce expression for 20 h at 22°C and 150rpm. After the expression is completed, collect the bacteria by low-temperature centrifugation and resuspend them in 100 mM KPi (pH 7.0) buffer. Add protease inhibitors PMSF and lysozyme with a final concentration of 1 mg / mL in sequence, and incubate at 35°C for 30 min. The bacterial solution after incubation was broken by a high-pressure homogenizer for 30 min to obtain a crude enzyme solution. The crude enzyme solution was centrifuged at 4°C and 12000 g for 60 min, and the supernatant was purified after filtration through a 0.22 μm cellulose acetate filter.

[0090] Protein purification was performed using nickel column affinity chromatography and ÄKTA protein purification system. First, the nickel column was equilibrated with loading and equilibration buffer (100 mM potassium phosphate buffer, 20 mM imidazole, pH 7.0), and then the crude enzyme solution was loaded at a loading flow rate of 2 mL / min. After equilibration again, gradient elution was performed with elution buffer (100 mM potassium phosphate buffer, 500 mM imidazole, pH=7.0) in different proportions. The eluate containing the target protein was collected and concentrated by ultrafiltration using an ultrafiltration tube (10 kDa molecular weight cutoff, AmiconUltra, Sigma Millipore), and the residual imidazole was replaced with 100 mM potassium phosphate buffer (pH 7.0) to obtain a pure enzyme solution, and protein expression was verified by SDS-PAGE. The verification results are shown in Figure 4 The protein concentration was determined using a modified BCA protein concentration assay kit (Shanghai Biotechnology). The purified protein was diluted to 0.5 mM and frozen in liquid nitrogen and stored at -80°C for later use.

[0091] 2. Initial results of pure enzyme of M3 mutant and optimization of reaction conditions

[0092] In order to verify the reaction activity of the pure enzyme of the M3 mutant, activity screening was performed under the conditions of model substrate 7a. 30 μL of pure enzyme of the M3 mutant (0.5 mM), 50 μL of immobilized GDH (36 U / mL), 80 μL of NADP + Sodium salt (3.75 mM, KPi solution), 80 μL Glucose (375 mM, KPi solution), 40 μL substrate 7a (75 mM, EtOH solution), 310 μL KPi (100 mM, pH 7), and then shaken at 800 rpm for 24 h at room temperature. LC-MS detection showed that the analytical yield of product 7b was 48%, ee The value is 99%, dr The value is greater than 19:1.

[0093] The reaction conditions were further optimized to improve the enzyme reaction activity. The purified M3 mutant protein concentration was diluted to 0.025 mM using the reaction buffer. Using 7a as the model substrate, the reaction conditions of the M3 mutant were optimized (including the type of cosolvent, reaction temperature, buffer type, buffer pH, and GDH concentration). GDH was purchased from Nantong Feiyu Biotechnology Co., Ltd. with a specification of 5 kU>70 U / mg. The relative activity of the test reaction was determined. ee Value and dr Value, where relative activity = product concentration under different conditions / product concentration under initial conditions. The results of the investigation on the optimization of reaction conditions are shown in Table 8.

[0094] Table 8 Results of optimization of reaction conditions for the M3 mutant

[0095] Solvent GDH (U / mL) Buffer Reaction time (h) Reaction temperature (℃) Relative activity ee (%) dr EtOH 3 KPi (pH 7.0) 24 25 1 99 >19:1 DMSO 3 KPi (pH 7.0) 24 25 1.31 99 >19:1 IPA 3 KPi (pH 7.0) 24 25 1.62 99 >19:1 EtOH 3 KPi (pH 7.0) 24 20 0.93 99 >19:1 EtOH 3 KPi (pH 7.0) 24 30 0.83 99 >19:1 EtOH 3 KPi (pH 7.0) 24 40 0.09 99 >19:1 EtOH 3 Citrate(pH 6.0) 24 25 0.56 99 >19:1 EtOH 3 KPi (pH 6.0) 24 25 0.46 99 >19:1 EtOH 3 KPi (pH 8.0) 24 25 1.29 99 >19:1 EtOH 3 Tris–HCl (pH 8.0) 24 25 1.31 99 >19:1 EtOH 3 Tris–HCl (pH 9.0) 24 25 1.91 99 >19:1 EtOH 1 KPi (pH 7.0) 24 25 0.94 99 >19:1 EtOH 5 KPi (pH 7.0) 24 25 0.90 99 >19:1 EtOH 3 KPi (pH 7.0) 48 25 1.17 99 >19:1 EtOH 3 KPi (pH 7.0) 72 25 1.35 99 >19:1

[0096] According to the optimized reaction conditions, the optimal reaction conditions were: 5 mM model substrate 7a, Tris-HCl (100 mM, pH 9.0) buffer, IPA as cosolvent, 25°C as reaction temperature, 3 U / mL GDH concentration, and 72 h reaction time. Under the optimal reaction conditions, the analytical yield of product 7b was 95%. ee The value is 99%, dr The value is greater than 19:1. The analytical yields before and after reaction optimization are shown in Table 9.

[0097] Table 9 Results of the M3 mutant before and after optimization of reaction conditions

[0098] Reaction conditions Analytical yield (%) ee (%) dr Before optimization 48 99 >19:1 After optimization 95 99 >19:1

[0099] The chiral separation structure of product 7b is as follows Figure 5 The chiral detection chromatographic column used was a chiral IG column - Daicel CHIRALPAK (5 μm filler, 4.6×250 mm), the mobile phase ratio was 20% acetonitrile / 80% water (containing 20 mM ammonium acetate), and the flow rate was 0.7 mL / min.

[0100] Example 4 Investigation of substrate suitability of M3 mutant

[0101] Under the optimal reaction conditions, the activity of other substrates was investigated using the M3 mutant. The results of substrate expansion are shown in Table 10. 2.0 mL of M3 mutant (0.5 mM), 2.0 mL of GDH (3 U / mL), 2.7 mL of NADP + Sodium salt (3.75 mM, Tris-HCl solution), 2.7 mL Glucose (375 mM, Tris-HCl solution), 1.3 mL 7a (5 mM, IPA solution), 9.3 mL Tris-HCl solution (100 mM, pH 9.0), react at 25°C for 72 h. After the reaction was monitored by LC-MS, the reaction was quenched with saturated ammonium chloride solution. The reaction mixture was extracted with dichloromethane, and the organic phase was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, the filtered organic phase was concentrated under reduced pressure, and purified by silica gel column chromatography. The separation yield was obtained by separating the substrate by column chromatography to measure the product weight, and the separation yield = the amount of product substance / the amount of substrate substance (hereinafter collectively referred to as the separation yield).

[0102] Table 10 Results of investigation on substrate expansion of M3 mutant

[0103]

[0104] Sequence Listing:

[0105] SEQ ID No.1

[0106] IR-F7 amino acid sequence

[0107] DEFINITION NAD(P)-dependent oxidoreductase [ Streptomyces ipomoeae ]

[0108] ACCESSION WP_009330409

[0109] VERSION WP_009330409.1

[0110] SOURCE Streptomyces ipomoeae

[0111] Sequence

[0112] MSNTKAAQAPVSVIGGLMGQALAAAFLKAGHPTTVWNRTAAKADQLVGEGAALAGSTADAIAASPLVVVCVTDYTAVRELLDPLAGALKGKVLVNLTTGTSTQARETAEWAADKEITYLDGAIMAIPPDIATDAAVLLYSGPKAAFD EHEATLRALGAAGTTYLDTDHGLSALYDMSLLGIMWGILNGFLHGAALLGTAEVKATTFAPLANTMINVVTEYVTAYAPQIDEGKYPAGDATMTVHQDALEHLAEESETLGINAEMPRFFKALVDRSVAAGHAESGYAALIEQFRKPAV

[0113] SEQ ID No.2

[0114] IR-B8 amino acid sequence

[0115] DEFINITION dehydrogenase, putative [ Labilithrix luteola ]

[0116] ACCESSION AKU97888

[0117] VERSION AKU97888.1

[0118] SOURCE Labilithrix luteola

[0119] Sequence

[0120] MARISILGAGRMGAALVTAFAKGGHAVTVWNRTASRAKPLEAVGARVALSLLDAIDADLVIDIVSDYEASAELVRNDDVSRALRDKTFLELASGTPKEAQRAAAWAEQNGIRYLDGAIMATPDFIGQPGCTILYSGPSAVFEEHRTILGAIAGNGLYLGSEIGHANVLDNAILVVLWGSVYGVLQGAAICEAERFSLDAFGSALEGAWPVVAPLLLSSIDRIGKRRWAADATTQSTLAPGYASVRHLLAMSKEYGIDAALPEALHRVFQRAVDAGHRDDDIAAAYQGMR

[0121] SEQ ID No.3

[0122] GDH amino acid sequence

[0123] DEFINITION glucose dehydrogenase Bacillus subtilis

[0124] ACCESSION BAA09024

[0125] VERSION BAA09024.1

[0126] SOURCE Bacillus subtilis

[0127] Sequence

[0128] ​MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0129] SEQ ID No.4

[0130] Amino acid sequence of mutant F7-I124V-P128C-T240L

[0131] MSNTKAAQAPVSVIGLGLMGQALAAAFLKAGHPTTVWNRTAAKADQLVGEGAALAGSTADAIAASPLVVVCVTDYTAVRELLDPLAGALKGKVLVNLTTGTSTQARETAEWAADKEITYLDGAVMAICPDIATDAAVLLYSGPKAAFDEHEATLRALGAAGTTYLDTDHGLSALYDMSLLGIMWGILNGFLHGAALLGTAEVKATTFAPLANTMINVVTEYVTAYAPQIDEGKYPAGDALMTVHQDALEHLAEESETLGINAEMPRFFKALVDRSVAAGHAESGYAALIEQFRKPAV。

Claims

1. An imine reductase mutant, characterized in that: It is derived from Streptomyces ipomoeae The imine reductase IR-F7 was used as the parent, and the mutants obtained by mutation were: The isoleucine at position 124 of the parent was mutated to valine to obtain the mutant F7-I124V; Among them, the amino acid sequence of imine reductase IR-F7 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 of F7-I124V as the parent: The proline at position 128 of the parent was mutated to cysteine ​​to obtain the mutant F7-I124V-P128C.

3. The imine reductase mutant according to claim 2, characterized in that: It is a mutant obtained by mutation of F7-I124V-P128C as the parent: The methionine at position 183 of the parent was mutated to phenylalanine to obtain the mutant F7-I124V-P128C-M183F; The tyrosine at position 221 of the parent was mutated to isoleucine to obtain the mutant F7-I124V-P128C-Y221I; The leucine at position 187 of the parent was mutated to phenylalanine to obtain the mutant F7-I124V-P128C-L187F; The threonine at position 240 of the parent was mutated to isoleucine to obtain the mutant F7-I124V-P128C-T240I; The threonine at position 240 of the parent was mutated to leucine to obtain the mutant F7-I124V-P128C-T240L; Among them, the amino acid sequence of the mutant F7-I124V-P128C-T240L is shown in SEQ ID NO.

4.

4. A DNA molecule, characterized in that: The DNA molecule encodes the imine reductase mutant according to any one of claims 1 to 3.

5. An expression vector, characterized in that: The expression vector contains the DNA molecule according to claim 4.

6. A host cell, characterized in that: The host cell contains the expression vector according to claim 5.

7. Use of the imine reductase mutant F7-I124V-P128C-T240L in the synthesis of polycyclic chiral nitrogen heterocyclic compounds; the polycyclic chiral nitrogen heterocyclic compounds have a structural formula as shown in Formula I or Formula II: ； in, n1=0, 1, 2, 3; n2=0, 1, 2, 3; X=CH2, NBn, NBz, NBoc; ； Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; R1=F, Cl, Br, I, CH3, CH3O; The synthetic substrate is shown in Formula III and Formula IV: 、 ； The amino acid sequence of the imine reductase mutant F7-I124V-P128C-T240L is shown in SEQ ID NO.

4.

8. A method for synthesizing a polycyclic chiral nitrogen heterocyclic compound, characterized in that: The method uses formula III or IV as a substrate and uses an imine reductase mutant F7-I124V-P128C-T240L as a biocatalyst to synthesize a polycyclic chiral nitrogen heterocyclic compound. The amino acid sequence of the imine reductase mutant F7-I124V-P128C-T240L is shown in SEQ ID NO.

4. Its synthetic route is shown in general formula I or general formula II: ； Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; X=CH2, NBn, NBz, NBoc; ； Among them, n1=0, 1, 2, 3; n2=0, 1, 2, 3; R1=F, Cl, Br, I, CH3, CH3O.

9. The method for synthesizing a polycyclic chiral nitrogen heterocyclic compound according to claim 8, characterized in that: The reaction system contains coenzyme, glucose and glucose dehydrogenase.

10. The method for synthesizing a polycyclic chiral nitrogen heterocyclic compound according to claim 9, characterized in that: The coenzyme is NADP + Salt.

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