Imine reductase mutant with improved enantioselectivity and application of imine reductase mutant in production of S-nicotine

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to an enantioselectivity-improved imine reductase mutant and application thereof in production of S-nicotine, and the enantioselectivity-improved imine reductase mutant is obtained by performing single-point or multi-point mutation on the 118th site, the 168th site, the 169th site, the 172th site, the 234th site and the 238th site of the imine reductase with the amino acid sequence as shown in SEQ ID NO.2. According to the present invention, the imine reductase derived from Pseudomonas sp. RC4D1, the accession number of which is WP213665450.1 in NCBI is subjected to mutation to obtain a series of mutants, such that the high enzyme activity of the wild type imine reductase is maintained while the enantioselectivity in the S-nicotine catalytic preparation reaction is improved; compared with the prior art, the method has the advantages of simple production process, mild reaction conditions, high selectivity of imine reductase, high optical purity of the product and better industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and specifically relates to an imine reductase mutant with improved enantioselectivity and its application in production S -Application of nicotine. Background Art

[0002] S - Nicotine, chemical name is 1-methyl-2-(3-pyridyl)pyrrolidine, common name is nicotine, mainly exists in Solanaceae plants, has strong physiological activity, in the field of medicine, S -Nicotine can be used to treat ADHD, depression, Alzheimer's disease and other diseases; in the agricultural field, it can be used as a high-efficiency, low-toxic spectrum pesticide; in addition, S -Nicotine is also an important raw material for cosmetics, functional health products, etc. There are three ways to obtain nicotine, namely extraction from tobacco leaves and other plants, chemical synthesis, and enzymatic synthesis. Direct extraction from tobacco leaves is the main way to obtain nicotine, but this method has cumbersome procedures, long cycles, and is easily interfered by impurities, and the purity is limited. Chemical synthesis requires splitting before obtaining S -nicotine, so the cost is relatively high, and there are also common problems of high consumption, high pollution and low conversion rate, which have many limitations in actual production.

[0003] Enzymatic synthesis of chiral compounds has attracted much attention due to its low cost and environmental friendliness. Imine reductase (IRED) is an effective catalyst for the preparation of various chiral amines. In 2010, Mitsukura and his colleagues first applied IRED to the synthesis of imines. They identified IRED from Streptomyces GF3587 and Streptomyces GF3546, respectively. R Selectivity and S Since then, a large number of naturally occurring IREDs have been identified and characterized for the synthesis of chiral amines. However, natural IREDs have been inconvenient for industrial application because their stereoselectivity does not meet production requirements or their selectivity varies with the structural differences of substrate molecules. In 2016, Turner's team discovered Amycolatopsis orientali The IREDs from the source exhibited mixed stereopreferences, which indicates that the stereoselectivity mechanism of IREDs is complex and must be studied on a case-by-case basis.

[0004] Therefore, it is of great significance to further explore imine reductases with high catalytic efficiency and use protein engineering technology to construct imine reductase mutants with better enantioselectivity to catalyze the synthesis of S-nicotine. Summary of the invention

[0005] Regarding the existing technology S-The defects of the nicotine synthesis process, the present invention provides an imine reductase mutant with improved enantioselectivity and its application in production S -Application in nicotine, the specific technical scheme is as follows: In a first aspect, the present invention provides an imine reductase mutant obtained by performing single-point or multi-point mutations at positions 118, 168, 169, 172, 234, and 238 of the imine reductase of the amino acid sequence as shown in SEQ ID NO.2.

[0006] Furthermore, the mutant form of the imine reductase mutant is one of the following mutant forms: (1) Alanine at position 118 mutated to serine; (2) Serine 168 mutated to arginine; (3) Alanine at position 169 mutated to threonine; (4) Cysteine ​​at position 172 mutated to tyrosine; (5) Alanine at position 234 mutated to serine; (6) Threonine at position 238 mutated to methionine; (7) Alanine at position 118 mutated to serine, and serine at position 168 mutated to arginine; (8) Alanine at position 118 mutated to serine, and alanine at position 169 mutated to threonine; (9) Alanine at position 169 mutated to threonine, and cysteine ​​at position 172 mutated to tyrosine; (10) Alanine at position 118 mutated to serine, alanine at position 169 mutated to threonine, and threonine at position 238 mutated to methionine; (11) Alanine at position 118 mutated to serine, alanine at position 169 mutated to threonine, alanine at position 234 mutated to serine, and threonine at position 238 mutated to methionine.

[0007] The present invention uses Pseudomonas Pseudomonas sp. RC4D1, and the imine reductase with the accession number WP_213665450.1 in NCBI are used as the basis for mutation. The enzyme has higher enzyme activity and lower enantioselectivity in the wild-type enzyme. The present invention mutates the residues around the imine reductase substrate to change the interaction between the enzyme molecule and the substrate. The obtained imine reductase mutant is effective in catalyzing the preparation of pseudooxynicotine S -In the reaction of nicotine, a good optical purity of even 99.9% can be obtained.

[0008] In a second aspect, the present invention provides a gene encoding the above-mentioned imine reductase mutant.

[0009] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned gene.

[0010] In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned gene.

[0011] In a fifth aspect, the present invention provides the above-mentioned imine reductase mutant, or the above-mentioned recombinant vector, or the above-mentioned genetically engineered bacteria in the preparation of S -Application of nicotine.

[0012] In a sixth aspect, the present invention provides a method for preparing S -nicotine method, in a coenzyme circulation system, using the crude enzyme solution, pure enzyme or immobilized enzyme of the above-mentioned imine reductase mutant, or the wet bacteria or whole cells of the above-mentioned genetically engineered bacteria as a catalyst, to carry out a reaction preparation S -Nicotine.

[0013] Furthermore, the coenzyme circulation system includes coenzymes, glucose and glucose dehydrogenase, and the coenzyme is NADP+ or NAD+.

[0014] Furthermore, the glucose dehydrogenase is derived from Bacillus cereus. Bc GDH (NCBI accession number EEL32290.1), from Bacillus megaterium Bm GDH (NCBI accession number WP_013055759.1) or from Bacillus amyloliquefaciens Ba One of the GDH (NCBI accession number: QKN90487.1).

[0015] Furthermore, in the reaction, the reaction temperature is 15-35° C., the pH value is 5.5-8.5, and the substrate concentration is 50-500 mM.

[0016] Furthermore, the catalytic reduction reaction is carried out in 100 mM phosphate buffer and the coenzyme concentration is 0.01-0.1 mM.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention is derived from Pseudomonas Pseudomonas sp. RC4D1 and the imine reductase with the accession number WP_213665450.1 in NCBI were mutated to obtain a series of mutants, which improved its catalytic activity while maintaining the high enzymatic activity of the wild-type imine reductase. S -Enantioselectivity in nicotine reaction. Compared with the prior art, the present invention has simple production process, mild reaction conditions, high selectivity of imine reductase, high optical purity of the product, and good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Imine reductase catalyzes the production of pseudooxynicotine S -Reaction equation of nicotine.

[0019] Figure 2 This is the achiral HPLC detection spectrum of the reaction solution.

[0020] Figure 3 for S -Achiral liquid chromatogram of nicotine standard.

[0021] Figure 4 This is the chiral HPLC detection spectrum of the reaction solution.

[0022] Figure 5 for S -Chiral HPLC detection spectrum of nicotine standard.

[0023] Figure 6 Prepared in Example 7 S -Reaction process diagram of nicotine. DETAILED DESCRIPTION

[0024] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0025] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions are carried out according to conventional experimental methods or according to the operating instructions recommended by the supplier. For gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0027] Reagents used in upstream genetic engineering operations: restriction endonucleases, PrimerSTAR, DNA polymerase, DNA ligase, and recombinase used in the examples of the present invention were purchased from TaKaRa; genome extraction kits, plasmid extraction kits, and DNA recovery and purification kits were purchased from Axygen; E. coliBL21 (DE3), plasmids, etc. were purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose electrophoresis reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primer synthesis and gene sequencing were completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The use of the above reagents refers to the product instructions.

[0028] The reagents used in the catalytic process: pseudooxynicotine (4-(methylamino)-1-(pyridin-3-yl)butanone), NADPH, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc. are all commercially available analytical grade.

[0029] The structural formula of pseudooxynicotine is shown in formula (1). S -The structural formula of nicotine is shown in formula (2).

[0030]

[0031] Preparation of the present invention S -The reaction equation of nicotine, such as Figure 1 shown.

[0032] The present invention uses high performance liquid chromatography (HPLC) to analyze the concentrations of substrates and products in the reaction solution to monitor the progress of the reaction.

[0033] The enzyme activity was analyzed by reverse system, and the analysis method was as follows: the chromatographic column model was YMC-Triart C18, 3 μm, 4.6×150 mm. The mobile phase was K 2 HPO 4 (10 mM): acetonitrile = 65:35, pH = 10; detection wavelength: 260 nm, flow rate: 1.0 mL / min, column temperature: 30°C. The peak conditions are as follows Figure 2 As shown in Figure 2, the peak of the product nicotine (standard product) is as follows: Figure 3 shown.

[0034] The chirality was analyzed by the forward system, and the analysis method was as follows: the chromatographic column model was Daicel OD-H column, 5 μm, 4.6×250 mm; the mobile phase was n-hexane: isopropanol: diethylamine = 98:2:0.1, the flow rate was 0.6 mL / min, the detection wavelength was 260 nm, and the column temperature was 25°C. The sample peaks were as follows Figure 4 As shown, the peak of nicotine (standard product) is as follows Figure 5 shown.

[0035] The enzyme activity was calculated as: Enzyme activity ( (1) In formula (1), k 1 : the slope of the nicotine calibration curve; b 1: The intercept of the nicotine standard curve; k 2 : dilution factor of the sample to be tested; V 1 : The volume of the reaction system, in L; V 2 : The volume of enzyme solution added to the reaction system, in L; k 3 : dilution multiple of enzyme solution; S 1 : Peak area of ​​nicotine, T: reaction time, unit min.

[0036] The amino acid sequence of the wild-type imine reductase used in the present invention is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.1.

[0037] SEQ ID NO.1: ATGCAAAGTGAAGTAACAGTTATAGGACTAGGGGCGATGGGTAGCGCGCTGGCGCACGTACTGTTGAGAGCGGGTAAACGTGTTACCGTTTGGAACCGCTCTCTCTCTCGTACTGAAAGCGCTGCGGGTGCGGGTGCCCACGTGTCTGAGACATTTATCAGCGCCATCTCCGCGAGTCCAGTGTGCCTGTTTTGTGTTGACAACTATGCAGTCACCAATGCACTGCTTACCGCAAATGATATCGAGGGCGTGTTGTCCGGCAAACTGTTAGTTCAGCTGTCCACCGGTGACCCGCAGGAGGCGCGTGATAGCGAGGAATGGGCATTGTCACGTGGTGCGGACTACCTGGATGCAGCGATTCTGGCGTTCCCGGCTCAAATGGGTACGCAAGAGGCGACCATTATTGTTAGCGGCGCGTTAGCCTCCTTTGAACGCGGTGCCTCGATCCTCAAGCTGCTGGCTCCGAACCTGTCGTACCTGGGCGACCGCGTTGGTGCTGCTTCGGCGCAGGATTGTGCGGTGGCCGCGTATTTCAGCGGCGCGCTGTTGGGCGCGTTACATGGTGCTCGTATTTGCGAAGTGGAAGGCCTGCCGGTGGACGAATACTGCAGCTTGCTGGCTGATATCTCCCCGGTTCTGGGCGGTGACATCCAGCACATGGGCGCTATGATTCATAGCGAGAGCTACAGCACCCCGCAGGCGAGCCTTAAGACGTGGGCGGCGGCGATCAGCCGTCTGGAACGTCACGCAAAAGCAGTCAAAATCAACCACGAATTTCCGGCATTCGCCTCTGCATTGTTCCGCTTGGGCGTTGATGCCGGCTATGGTAGCGAGGAGGTTGCAGCGCTGATTAAGGTGTTACGTGCGGTCAGCGAGGTGCGTTAA SEQ ID NO.2: MQSEVTVIGLGAMGSALAHVLLRAGKRVTVWNRSLSRTESAAGAGAHVSETFISAISASPVCLFCVDNYAVTNALLTANDIEGVLSGKLLVQLSTGDPQEARDSEEWALSRGADYLDAAILAFPAQMGTQEATIIVSGALASFERGA SILKLLAPNLSYLGDRVGAASAQDCAVAAYFSGALLGALHGARICEVEGLPVDEYCSLLADISPVLGGDIQHMGAMIHSESYSTPQASLKTWAAAISRLERHAKAVKINHEFPAFASALFRLGVDAGYGSEEVAALIKVLRAVSEVR Example 1 Construction of wild enzyme engineering bacteria Search the National Coalition Building Institute (NCBI) database for imine reductase and select the enzyme from Pseudomonas Pseudomonas sp. RC4D1( Ps IRED, database NCBI accession number WP_213665450.1, base sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2); the amino acid sequence was converted into nucleotide sequence by codon optimization. The nucleotide sequence was fully synthesized by chemical method (Yixin Biotechnology) and integrated into the expression vector pET-28a(+) multiple cloning site; finally, the constructed plasmid was introduced into Escherichia coli BL21 (DE3) host cells to construct wild imine reductase engineering bacteria.

[0038] Example 2 Construction of imine reductase mutants 1. Activation of engineered bacteria and plasmid extraction The engineered bacteria constructed in Example 1 were activated and cultured using LB medium.

[0039] The LB medium formula is: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, dissolved in deionized water and fixed to volume, sterilized at 121℃ for 20 min, and set aside. The solid medium is LB medium with 2% (mass percentage) agar added.

[0040] The preserved engineered bacteria glycerol tube was inoculated into a test tube containing 10 mL LB medium and cultured at 37°C and 200 rpm for 12 hours. After obtaining the cultured bacteria, plasmids were extracted according to the operating instructions of the Axygen plasmid extraction kit. The obtained plasmids can be directly used for point mutation or stored at -80°C for a long time.

[0041] 2. Gene site-directed mutagenesis Gene mutation uses the whole plasmid PCR method. When only a small range of mutation is required, the mutation can be designed in the upstream and downstream primers, and PCR can be performed using the plasmid as a template to obtain the target plasmid.

[0042] Table 1 PCR amplification system

[0043] PCR amplification program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55-58°C (adjusted according to primer Tm value) for 15 s, extension at 72°C for 75 s (adjusted according to gene length, 10 s / 1 kb), for a total of 30 cycles; extension at 72°C for another 10 min; and storage at 4°C.

[0044] After PCR amplification, the amplified product was detected by 0.9% (mass percentage) agarose gel electrophoresis. The results showed that the amplified product was a single band. The amplified product was purified and recovered using a DNA recovery kit. For specific steps, refer to the instructions of the purification kit.

[0045] The mutation points of the mutants and the primers designed according to the mutation points are shown in Table 2.

[0046] Table 2 Mutation points and primers

[0047] 3. Construction of mutant engineered bacteria The purified gene fragments were used Dpn I digestion to remove the template, and then use the recombinase to recombine; the recombinant product is transformed into E.coil In BL21 (DE3) competent cells, plate and pick single colonies to LB liquid culture, identify the successfully constructed positive transformants by PCR, and verify the correctness of the mutation site by sequencing. After verification, add sterile glycerol with a final concentration of 20% (volume percentage), mark and store at -80℃ for later use.

[0048] Example 3 Cultivation of Imine Reductase Bacteria and Preparation of Enzyme Powder After the engineered bacteria containing the imine reductase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 10 h. A 2% inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 μg / mL Kan and cultured at 37°C with shaking until the OD 600When the pH reaches about 0.6-0.8, IPTG is added to a final concentration of 0.25 mM, and the culture is induced at 18°C ​​for 18 h. After the culture is completed, the culture solution is centrifuged at 4000 rpm for 10 min, the supernatant is discarded, and the bacteria are collected. The collected bacteria are resuspended in 2 volumes of 100 mM Tris-HCl buffer at pH 7.5, ultrasonically broken, and centrifuged at 12000 rpm at 4°C for 10 min to remove the precipitate. The obtained supernatant solution is freeze-dried to obtain imine reductase enzyme powder.

[0049] Example 4 Cultivation of glucose dehydrogenase bacteria and preparation of enzyme powder After the engineered bacteria containing the glucose dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 8 h. A 2% inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 μg / mL Kan and cultured at 37°C with shaking until the OD 600 When the pH reaches about 0.6-0.8, add IPTG to a final concentration of 0.5 mM, and induce culture at 18°C ​​for 16-18 h. After the culture is completed, centrifuge the culture solution at 4000 rpm for 10 min, discard the supernatant, and collect the bacteria. Resuspend the collected bacteria in 2 volumes of 100 mM Tris-HCl buffer at pH 7.5, ultrasonically disrupt, centrifuge the disrupted solution at 12000 rpm at 4°C for 10 min to remove the precipitate, and freeze-dry the obtained supernatant solution to obtain glucose dehydrogenase enzyme powder.

[0050] Example 5 Purification of Imine Reductase and Its Mutants The crude enzyme solution of imine reductase and its mutants was obtained in the manner of Example 3. The imine reductase had a 6×His fusion tag at the N-terminus. Since the imidazole group of histidine could chelate with Ni ions, a Ni-NTA protein purification column was used for protein purification.

[0051] The specific steps are as follows: Pretreatment of protein samples and Ni-NTA protein purification column: The obtained imine reductase crude enzyme solution was filtered using a 0.22 μm filter membrane to further remove impurities. The Ni-NTA protein purification column was rinsed with 10 column volumes of ultrapure water to ensure that the 20% (volume percentage) ethanol in the column was rinsed clean, and the Ni-NTA protein purification column was equilibrated with 10 column volumes of 25 mM imidazole solution.

[0052] Protein purification and elution: Repeat the filtration of the imine reductase protein sample twice. Rinse the Ni-NTA affinity column with 10 column volumes of 25 mM imidazole solution to remove unbound impurities. Rinse the Ni-NTA affinity column with 10 column volumes of 250 mM imidazole to elute the target protein, collect the eluted target protein and store it on ice.

[0053] Protein ultrafiltration desalting: The collected target protein was concentrated by centrifugation at 4000 rpm for 30 min at 4°C using a Millipore ultrafiltration tube with a molecular weight cutoff of 10 kDa. After concentration, it was washed and replaced with 0.1 M phosphate buffer to remove imidazole, and finally concentrated to about 1 mL, and glycerol with a final concentration of 20% (v / v) was added. The protein concentration was determined by the Bradford method and stored in a -80°C refrigerator for later use.

[0054] Example 6 Enzyme activity of imine reductase and its mutants and ee Value determination Imine reductase was obtained according to the method of Example 5 Ps The wild type and mutant pure enzymes of IRED. A 400 μL reaction system was established, in which the final concentration of imine reductase was 0.1 mg / mL, the concentration of substrate pseudooxynicotine was 10 mM, and the final concentration of coenzyme NADPH was 10 mM. The reaction was carried out in 0.1 M phosphate buffer, pH 6.5. The reaction temperature and speed were controlled by a shaker. The reaction solution was reacted at 25°C and 220 rpm for 16 h, and then 400 μL of n-hexane was added, oscillated and extracted. After centrifugation, the supernatant was taken for chiral analysis, and the relative enzyme activity and ee The results are shown in Table 3, among which the enzyme activities of mutants A118S, S168R, A169T, C172Y, A234S, T238M, A118S / S168R, A118S / A169T, A169T / C172Y, A118S / A169T / T238M, A118S / A169T / T238M / A234S and ee The values ​​were increased compared with the wild type.

[0055] Table 3 Specific enzyme activity of imine reductase and its mutants catalyzing pseudooxynicotine ee value

[0056] Example 7 Using imine reductase and glucose dehydrogenase Bc GDH preparation S -Nicotine Imine reductase and glucose dehydrogenase were obtained according to the method of Example 3 and Example 4 BcGDH enzyme powder. Use pH7.0, 0.1 M Tris-HCl solution to prepare a mixed solution of 0.5 M substrate pseudooxynicotine and 0.5 mM coenzyme NADP+ for later use. Add 20 mL pH7.5, 0.1 M Tris-HCl buffer to a 50 mL three-necked flask, then add 0.1 g imine reductase, 0.02 g Bc GDH, and 30 mL of the pre-prepared mixed solution of substrate and coenzyme. The reaction temperature was controlled at 20°C by water bath, magnetic stirring was applied, and samples were taken at regular intervals. The concentration and ee Value, where the reaction progress is as Figure 6 The detection results after 8 hours of reaction are shown in Table 4.

[0057] Table 4 Preparation of imine reductase and its mutants S -Nicotine yield and ee value

[0058] Example 8 Using imine reductase and glucose dehydrogenase Bm GDH preparation S -Nicotine Imine reductase and glucose dehydrogenase were obtained according to the method of Example 3 and Example 4 Bm GDH enzyme powder. Use pH 8.0, 0.1 M Tris-HCl solution to prepare a mixed solution of 0.3 M substrate pseudooxynicotine and 0.3 mM coenzyme NAP+ for later use. Add 20 mL pH 8.0, 0.1 M Tris-HCl buffer to a 50 mL three-necked flask, then add 0.1 g imine reductase, 0.02 g Bm GDH, and 30 mL of the pre-prepared substrate and coenzyme mixed solution. The reaction temperature was controlled at 25°C by water bath, magnetic stirring was used, and the concentration and ee The results are shown in Table 5.

[0059] Table 5 Preparation of imine reductase and its mutants S -Nicotine yield and ee value

[0060] Example 9 Using imine reductase and glucose dehydrogenase Ba GDH preparation S -Nicotine Imine reductase and glucose dehydrogenase were obtained according to the method of Example 3 and Example 4 BaGDH enzyme powder. Use pH 8.5, 0.1 M Tris-HCl solution to prepare a mixed solution of 0.4 M substrate pseudooxynicotine and 0.4 mM coenzyme NAD+ for later use. Add 20 mL pH 6.5, 0.1 M Tris-HCl buffer to a 50 mL three-necked flask, then add 0.1 g imine reductase, 0.02 g Ba GDH, and 30 mL of the pre-prepared substrate and coenzyme mixed solution. The reaction temperature was controlled at 30°C by water bath, and magnetic stirring was performed. After 8 h of reaction, the concentration and ee The results are shown in Table 6.

[0061] Table 6 Preparation of imine reductase and its mutants S -Nicotine yield and ee value

Claims

1. An imine reductase mutant, characterized in that: The amino acid sequence is obtained by performing single-point or multi-point mutations at positions 118, 168, 169, 172, 234 and 238 of the imine reductase shown in SEQ ID NO.

2.

2. The imine reductase mutant according to claim 1, characterized in that The mutant form of the imine reductase mutant is one of the following mutant forms: (1) Alanine at position 118 mutated to serine; (2) Serine 168 mutated to arginine; (3) Alanine at position 169 mutated to threonine; (4) Cysteine ​​at position 172 mutated to tyrosine; (5) Alanine at position 234 mutated to serine; (6) Threonine at position 238 mutated to methionine; (7) Alanine at position 118 mutated to serine, and serine at position 168 mutated to arginine; (8) Alanine at position 118 mutated to serine, and alanine at position 169 mutated to threonine; (9) Alanine at position 169 mutated to threonine, and cysteine ​​at position 172 mutated to tyrosine; (10) Alanine at position 118 mutated to serine, alanine at position 169 mutated to threonine, and threonine at position 238 mutated to methionine; (11) Alanine at position 118 mutated to serine, alanine at position 169 mutated to threonine, alanine at position 234 mutated to serine, and threonine at position 238 mutated to methionine.

3. A gene encoding the imine reductase mutant according to claim 1.

4. A recombinant vector, characterized in that: The recombinant vector comprises the gene according to claim 3.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the gene as claimed in claim 3.

6. The imine reductase mutant according to claim 1, or the recombinant vector according to claim 4, or the genetically engineered bacterium according to claim 5 in the preparation of S -Application of nicotine.

7. Preparation S -nicotine method, characterized in that In a coenzyme circulation system, the crude enzyme solution, pure enzyme or immobilized enzyme of the imine reductase mutant as claimed in claim 1, or the wet bacteria or whole cells of the genetically engineered bacteria as claimed in claim 5 are used as catalysts to prepare the reaction. S -Nicotine.

8. The method according to claim 7, characterized in that The coenzyme circulation system includes coenzymes, glucose and glucose dehydrogenase, and the coenzyme is NADP+ or NAD+.

9. The method according to claim 7, characterized in that In the reaction, the reaction temperature is 15-35° C., the pH value is 5.5-8.5, and the substrate concentration is 50-500 mM.

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