Imine reductase mutant and its application and application method

Abstract

The present invention belongs to the technical field of biocatalytic organic synthesis, and particularly relates to an imine reductase mutant and its application and application method. The present invention is that the amino acid sequence of the imine reductase mutant is an amino acid sequence mutated on the amino acid sequence shown in SEQ ID NO.1, and the optional mutation site includes one of the following sites: the 44th G mutates to V or the 89th L mutates to V or the 44th G and the 89th L mutate to V at the same time. The imine reductase mutant of the present invention combines with glucose dehydrogenase, takes β-nicotinamide adenine dinucleotide disodium salt as a coenzyme, takes 5-(3,4-dihydro-2H-pyrrole-5-yl)-2-methylpyridine as a substrate, and prepares the intermediate (S)-2-methyl-5-(pyrrole-2-yl)pyridine by asymmetric hydrogenation reduction, and its chemical conversion rate can reach more than 99.5%, and ee% can reach 99.6%, which lays a production foundation for the industrialization of (S)-2-methyl-5-(1-methylpyrrole-2-yl)pyridine, and has good industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalytic organic synthesis, and particularly relates to an imine reductase mutant and an application and an application method thereof. Background Art

[0002] Neurodegenerative diseases are one of the major threats to human health. With the continuous deepening of research on such diseases, some scholars believe that nicotine acetylcholine receptors (nAChR) in neurons are important targets for drug treatment (Neuropharmacology, 1995, 34, 563). Many studies have confirmed that natural alkaloid nicotine has a certain effect on alleviating and treating Parkinson's disease, Alzheimer's disease and Tourette syndrome. However, it has toxic side effects on the cardiovascular system and digestive system, which greatly limits its clinical application (ChemEng. News 2000, 78, 23-26). Existing literature (J.Med.Chem.1997,40,4169; J.Pharm.Exp.Ther.2000,292,461) summarizes the structure-activity relationship of nAChR ligands through systematic research. As early as the end of the 20th century, some new nicotine analogs have been discovered and used for clinical treatment of Parkinson's disease (J.org.Chem.1999,64,5299). Dukat et al. (Eur.J.Med.Chem.1999,34,31) summarized and reported that the affinity of substituted nicotine to nAChR receptors is related to the lipophilicity of the 6-substituent. Related studies further show that as the size of the substituent increases, the affinity decreases, that is, the affinity of nicotine analogs is determined by the lipophilicity of the 6-substituent and is also related to the size of the substituent. Among them, the 6-substituent of 6-methylnicotine has the smallest volume, strong lipophilicity, and strong affinity with nAChR receptors, and has a good prospect for pharmaceutical use. The existing technologies are basically focused on the synthesis of racemates, and there is no report on the synthesis of single configuration.

[0003] Finding imine reductases from different biological sources and using protein engineering methods to modify the enzyme molecules to obtain more efficient imine reductases is the direction of efforts to achieve the industrial production of nicotine's key chiral intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine by enzymatic method. Summary of the invention

[0004] The object of the present invention is an imine reductase mutant, which can reduce and hydrogenate 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine and convert it into a single chiral intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine, wherein the chemical conversion rate can reach more than 99.5%, and the ee% can reach 99.6%, thereby laying a production foundation for the industrialization of (S)-2-methyl-5-(1-methylpyrrol-2-yl)pyridine and having good industrial application value.

[0005] The invention mutates the imine reductase by using a random mutation molecular biology method on the basis of the wild-type imine reductase shown in the amino acid sequence SEQ NO.1 and the nucleotide sequence SEQ NO.5, thereby changing the amino acid sequence of the enzyme and realizing the change of the enzyme structure and function, and then obtains three imine reductase mutants in which the 44th G is mutated to V, the 89th L is mutated to V, or the 44th G and the 89th L are mutated to V on the basis of the amino acid of SEQ NO.1 by a directed screening method.

[0006] Specifically, the amino acid sequence of the imine reductase mutant of the present invention is shown in SEQ NO.2, SEQ NO.3 or SEQ NO.4.

[0007] Specifically, the nucleotide sequence of the imine reductase mutant SEQ NO.2 of the present invention is shown in SEQ NO.6.

[0008] Specifically, the nucleotide sequence of the imine reductase mutant SEQ NO.3 of the present invention is shown in SEQ NO.7.

[0009] Specifically, the nucleotide sequence of the imine reductase mutant SEQ NO.4 of the present invention is shown in SEQ NO.8.

[0010] The present invention also provides a recombinant plasmid comprising a gene encoding the imine reductase mutant described in the above amino acid sequence such as SEQ NO.2, SEQ NO.3 or SEQ NO.4.

[0011] The present invention also provides a host cell comprising the above recombinant plasmid. The host cell is selected from a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is an Escherichia coli BL21 (DE3) cell.

[0012] The present invention also provides the use of the imine reductase mutant in the synthesis of the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine.

[0013] The application method is: combining the imine reductase mutant with glucose dehydrogenase, using β-nicotinamide adenine dinucleotide disodium salt as a coenzyme and 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine as a substrate, and asymmetric hydrogenation reduction to prepare the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine.

[0014] Preferably, the pH value of the reaction process of the imine reductase mutant to prepare the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine is 6.8 to 8.0;

[0015] Preferably, the pH value is 7.2 to 7.6, and an 8% sodium hydroxide aqueous solution is used as a pH regulator to adjust the pH value during the reaction. When the pH value during the reaction is lower than 6.8 or higher than 8.0, the reaction conversion rate and reaction speed are significantly reduced.

[0016] Preferably, the reaction temperature of the reaction process of preparing the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine by the imine reductase mutant is controlled at 15 to 35°C;

[0017] Further preferably, the reaction temperature ranges from 25 to 30° C. When the temperature is lower than 15° C. or higher than 35° C., the reaction conversion rate and reaction rate are significantly reduced.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The imine reductase mutant of the present invention is used to catalyze the asymmetric hydrogenation reduction of 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine. Compared with the wild-type imine reductase, the enzyme activity is increased by more than 10 times, and the mutant has a very high chemical conversion rate and stereoselectivity.

[0020] 2) When the imine reductase mutant of the present invention is used to prepare the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine, the chemical conversion rate can reach more than 99.5%, and the ee% can reach 99.6%, which lays a production foundation for the industrialization of (S)-2-methyl-5-(1-methylpyrrol-2-yl)pyridine and has good industrial application value. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the embodiments.

[0022] Embodiment 1: Obtain wild-type imine reductase parent recombinant plasmid of Actinomadura sp. CNU-125 strain:

[0023] Through the NCBI Genbank nucleic acid database, the full-length gene was optimized by codon optimization and commissioned to a service provider to be artificially synthesized into the pET21a (+) expression plasmid, transformed into Escherichia coli BL21 (DE3) competent cells, and spread on LB agar plates containing 100 mg / L ampicillin, and cultured at 37°C for 24 hours. Several single colonies were selected and cultured in LB medium containing 100 mg / L ampicillin at 37°C for 24 hours, and then the recombinant plasmid was extracted using a plasmid mini-extraction kit, and PCR and sequencing verification were performed to obtain the imine reductase recombinant plasmid.

[0024] Embodiment 2: The wild-type imine reductase parent gene was randomly mutated.

[0025] According to the contents described in Example 1, the recombinant plasmid containing the imine reductase parental coding gene of the Actinomadurasp strain was used as a template, and primers at both ends were designed and synthesized using Primer5.0 (Table 1) according to the imine reductase parental coding gene of the Actinomadurasp strain, and error-prone PCR technology (material concentrations are shown in Table 2, and reaction conditions are shown in Table 3) was used to obtain linear gene fragments with a large number of base mutations, and then these PCR products and the pET21a (+) expression plasmid were respectively subjected to restriction digestion, gel recovery, ligation and transformation of Escherichia coli BL21 (DE3) competent cells, spread on LB agar plates containing 100 mg / L ampicillin, and cultured at 37° C. for 24 hours.

[0026] Table 1 Random mutagenesis primer sequences

[0027]

[0028] Table 2. 50 μL error-prone PCR material system

[0029]

[0030] Table 3. Error-prone PCR reaction conditions

[0031]

[0032] Embodiment 3: Cloning and expression of imine reductase mutants

[0033] Compatible restriction enzyme cleavage sites were designed at the 5' and 3' ends of the imine reductase gene to facilitate the cloning, expression and identification of imine reductase mutants. The target gene and pET21a (+) can be simultaneously digested and DNA gel-cut and recovered using Nde I and Xho I restriction enzymes, respectively. The recovered target gene and the larger fragment of the plasmid are ligated using T4 DNA ligase, and the continuous product is transformed into Escherichia coli BL21 (DE3) competent cells, and then the transformed competent cells are spread on LB agar plates containing 100 mg / L ampicillin and cultured at 37°C for 24 hours. A single bacterial cell grown in the above culture plate is selected and cultured in LB liquid culture medium containing 100 mg / L ampicillin at 37°C for 24 hours, and the bacterial cell is subjected to plasmid extraction, PCR identification and double enzyme digestion identification, and the correct recombinant plasmid is renamed, and then the subsequent induced expression is performed. The above bacterial solution was transferred to 500 mL of LB liquid culture medium containing 100 mg / L ampicillin and cultured at 37°C with shaking. When OD600 = 0.6-0.8, IPTG was added for induction. The expression was induced at 20-25°C for 18-24 hours. The bacterial solution was removed, and then centrifuged in a high-speed centrifuge for 20-30 minutes. The bacteria were collected and frozen at -20°C for later use.

[0034] Embodiment 4: Preliminary screening of imine reductase mutants

[0035] According to the contents of Example 2 and Example 3, the monoclonal strain on the above LB agar medium was inoculated into a 96-deep-well plate, 1 mL of 100 mg / L ampicillin solution was added to each well in advance, and the culture was shaken at 37°C for 4 hours. The inducer IPTG was added and the culture was induced at 20-25°C for 12-16 hours. The cells were centrifuged in a high-speed centrifuge for 20-30 minutes to collect the bacteria. Then the bacteria were added to a pH 7 phosphate buffered saline solution (concentration 100 mM), and the bacteria were crushed using a high-throughput ultrasonic cell crusher, with a crushing working time of 3 seconds, an interval of 3 seconds, and a temperature of 0-4°C. The supernatant obtained by high-speed centrifugation was then the crude enzyme solution of the imine reductase mutant. The activity was initially screened using an ELISA instrument. Add 20uL of 2mmol / L reduced nicotinamide adenine dinucleotide (NADH), 100uL of 100mM phosphate buffer solution (pH 7), 20uL of enzyme solution, and 20uL of 20mmol / L 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine solution to a 96-well plate, and detect the change of A340 every 15 seconds within 5 minutes.

[0036] Note: Enzyme activity calculation formula:

[0037] Enzyme activity (U / mL) = (ΔA×V1×1000) / (6220×t×V2)

[0038] ΔA: absorbance change within t;

[0039] V1: total volume of reaction system, mL;

[0040] V2: volume of enzyme solution added, mL;

[0041] 6220: molar extinction coefficient, L / mol / cm;

[0042] t: detection time.

[0043] Embodiment 5: Rescreening of Imine Reductase Mutants

[0044] Preparation of imine reductase mutant enzyme solution: The mutant with high enzyme activity in Example 4 was inoculated at 0.1% inoculum into 500 mL LB medium containing 100 mg / L ampicillin solution and cultured at 37° C. for 12-18 hours with shaking, and the inducer IPTG was added and induced at 20-25° C. for 16-24 hours. The cells were collected after high-speed centrifugation, and the cells were redispersed in 100 mM phosphate buffer solution (pH 7), and homogenized using a cell homogenizer at 600-800 bar and a temperature of 0-5° C. to obtain a crude imine reductase mutant enzyme solution.

[0045] Rescreening of crude enzyme solution, catalytic reduction of 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine: add 0.1 g of 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine, 2 mL of purified water, 2 mg of β-nicotinamide adenine dinucleotide phosphate disodium salt, 0.5 mL of imine reductase mutant enzyme crude solution, and 0.5 mL of glucose dehydrogenase crude solution into a 10 mL three-necked flask, control the reaction pH to 7.0-7.5, react at 27°C for 24-48 hours, analyze the reaction conversion rate and ee%, and the screening results are shown in Table 4.

[0046] Table 4 Comparison of the activity of the parent imine reductase and the mutants in preparing the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine

[0047]

[0048] Note: The enzyme amount refers to the wet weight of the recombinant cells of imine reductase required to transform 1g of substrate.

[0049] Embodiment 6: Determination of the catalytic conversion rate of imine reductase

[0050] The reaction solution of the reaction system was diluted 10 times with 70% acetonitrile aqueous solution, the protein was removed by precipitation, and after membrane filtration, it was diluted 10 times with purified water for sample injection.

[0051] Chromatographic conditions were: Agilent 1200 HPLC;

[0052] Column: XBridge C18, 4.6 x 150 mm 3.5 μM;

[0053] Mobile phase A: Accurately weigh 1.5 g of glacial acetic acid into a 25 ml volumetric flask, dilute it with purified water and add it to 900 ml of purified water. Then adjust the pH to 10.0 with 25% concentrated ammonia water, and then make up the solution to 1000 ml with purified water.

[0054] Mobile phase B: acetonitrile

[0055] Chromatographic conditions:

[0056]

[0057]

[0058] Conversion rate calculation formula: Conversion rate (%) = A P / (A P +A S )*100

[0059] A P : Product (S)-2-methyl-5-(pyrrol-2-yl)pyridine peak area

[0060] As: Peak area of ​​raw material 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine

[0061] Embodiment 7: Determination of Optical Purity of Substrate Catalyzed by Imine Reductase

[0062] The reaction product of Example 5 was post-processed and extracted, then concentrated to dryness, and a sample was diluted with pure ethanol to prepare a 2 mg / mL solution for injection.

[0063] Instrument: Agilent 1200 HPLC

[0064] Chromatographic column: CHIRALPAK IC 250*4.6mm 5μm

[0065] Mobile phase A: Accurately transfer 2.0 mL of diethylamine to 980 mL of n-hexane and 20 mL of isopropanol, mix well, and degas.

[0066] Mobile phase B: n-hexane

[0067] Chromatographic conditions:

[0068]

[0069] Note: The calculation formula of ee% of S-type product is: ee(%)=(A S -A R ) / ((A S+A R )*100

[0070] A S : Product (S)-2-methyl-5-(pyrrol-2-yl)pyridine peak area

[0071] A R : Enantiomer (R)-2-methyl-5-(pyrrol-2-yl)pyridine peak area

[0072] Embodiment 8: Preparation of Imine Reductase Mutant Enzyme Solution

[0073] The imine reductase mutant strain screened in Example 5 was inoculated at 0.01% in 10 bottles of 500 mL bottles, LB medium containing 100 mg / L ampicillin was added to the bottles in advance, and cultured at 37°C for 12-16 hours with shaking, and a certain amount of inducer propyl-β-D-thiogalactoside (IPTG, final concentration of 0.1 mM) was added, and the induction culture was continued at 20-25°C for 16-24 hours, and the cells were collected by high-speed centrifugation. The obtained 50 grams of cells were resuspended in 100 grams of purified water, and the crude enzyme solution of imine reductase mutants was homogenized and crushed.

[0074] Embodiment 9: The imine reductase mutant shown in SEQ ID NO.4 is used in reducing substrates to prepare the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine.

[0075] Add 50g of 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine to a 500mL reaction bottle, the solvent is 150mL of pure water, 123g of monohydrate glucose, 90mL of imine reductase mutant enzyme solution (the weight ratio of bacterial cells is 0.6), 90mL of glucose dehydrogenase solution (the weight ratio of bacterial cells is 0.6) and 0.66g of β-nicotinamide adenine dinucleotide phosphate disodium salt, adjust the temperature to 27°C, monitor the reaction pH change in real time, and use 8% sodium hydroxide aqueous solution to control the reaction pH between 7.2-7.5. The conversion rate is 88.3% for 24 hours and 99.5% for 48 hours. After the reaction is completed, adjust the pH to 2-3 with acid, then add 5g of activated carbon and 5g of diatomaceous earth, filter, adjust the filtrate to pH 13, then extract with methyl tert-butyl, combine the organic phases and concentrate to obtain 48.2g of the intermediate product with an ee% of 99.6%.

[0076] The amino acid and nucleotide sequences of the glucose dehydrogenase are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0077] The amino acid and nucleotide sequences involved in the present invention are as follows:

[0078] SEQ ID NO.1 (imine reductase amino acid sequence):

[0079] MKTPVTVLGLGAMGRALVTALLDAGRPVTVWNRTPGKADELAGAVEAASVHDAVTAGGIVVVCLYDHASVHETLDPVAAGLRGRAVVNLTTTTPAEARELAEWADGHGIEYLDGAIMATPPMIGAPGAQILYSGSRAVFDGHR EALDLWATSVYDGADAGMASLFDLAILSGMYPLFAGFLHGAAMVRAEGVPAAEFAERAAPFLAAMTGSFAGIARTVDGGGYDVPGQSLDWTAGALEAIGRASREQGVDPVPVDMVGALVRRQIDEGYGSEDMSRLIESMTGDAV

[0080] SEQ ID NO.2 (amino acid sequence of imine reductase mutant G43V):

[0081] MKTPVTVLGLGAMGRALVTALLDAGRPVTVWNRTPGKADELAVAVEAASVHDAVTAGGIVVVCLYDHASVHETLDPVAAGLRGRAVVNLTTTTPAEARELAEWADGHGIEYLDGAIMATPPMIGAPGAQILYSGSRAVFDGHR EALDLWATSVYDGADAGMASLFDLAILSGMYPLFAGFLHGAAMVRAEGVPAAEFAERAAPFLAAMTGSFAGIARTVDGGGYDVPGQSLDWTAGALEAIGRASREQGVDPVPVDMVGALVRRQIDEGYGSEDMSRLIESMTGDAV

[0082] SEQ ID NO.3 (amino acid sequence of imine reductase mutant L89V):

[0083] MKTPVTVLGLGAMGRALVTALLDAGRPVTVWNRTPGKADELAGAVEAASVHDAVTAGGIVVVCLYDHASVHETLDPVAAGLRGRAVVNVTTTTPAEARELAEWADGHGIEYLDGAIMATPPMIGAPGAQILYSGSRAVFDGHREALDLWATSVYDGADAGMASLFDLAILSGMYPLFAGFLHGAAMVRAEGVPAAEFAERAAPFLAAMTGSFAGIARTVDGGGYDVPGQSLDWTAGALEAIGRASREQGVDPVPVDMVGALVRRQIDEGYGSEDMSRLIESMTGDAV

[0084] SEQ ID NO.4 (Amino acid sequence of imine reductase mutant G43V-L89V):

[0085] MKTPVTVLGLGAMGRALVTALLDAGRPVTVWNRTPGKADELAVAVEAASVHDAVTAGGIVVVCLYDHASVHETLDPVAAGLRGRAVVNVTTTTPAEARELAEWADGHGIEYLDGAIMATPPMIGAPGAQILYSGSRAVFDGHREALDLWATSVYDGADAGMASLFDLAILSGMYPLFAGFLHGAAMVRAEGVPAAEFAERAAPFLAAMTGSFAGIARTVDGGGYDVPGQSLDWTAGALEAIGRASREQGVDPVPVDMVGALVRRQIDEGYGSEDMSRLIESMTGDAV

[0086] SEQ ID NO.5 (Imine reductase nucleotide sequence)

[0087] atgaagaccc ccgtgaccgt gctcgggctg ggtgcgatgg gacgtgcgct ggtcaccgcgctgctcgacg cggggcggcc ggtcaccgtg tggaaccgga cgcccgggaa ggcggacgaa ctggcgggcgcggtcgaggc cgcgagcgtc cacgacgcgg tgaccgcggg cgggatcgtg gtcgtgtgcc tctacgaccacgcctcggtg cacgagacac tcgacccggt ggccgccggg ctgcgcggac gggcggtggt gaacctcacgaccacgacgc ccgccgaggc acgggagctc gcggagtggg cggacggcca cggaatcgag tacctcgacggagcgatcat ggcgacgccg ccgatgatcg gtgctccggg cgcgcagatc ctctacagcg ggtcgcgtgcggtgttcgat gggcatcgcg aggcgctcga cctgtgggcg acgtccgtct acgacggtgc ggacgccggaatggcgtcgc tgttcgacct cgcgatcctt tcgggcatgt atccgctgtt cgccggattc ctgcacggcgccgcgatggt ccgcgccgag ggcgtcccgg cggccgagtt cgcggagcgc gccgcgcctt tcctcgccgcgatgaccggg tcgttcgccg gcatcgcgcg aaccgtggac ggcggcggct acgacgtgcc ggggcagagcctcgactgga cggccggcgc gctggaggcc atcggccgtg cgagccgcga gcagggcgtc gatccggttccggtcgacat ggtgggggcg ctcgtccggc ggcagatcga cgaagggtac gggagcgaag atatgtcccggctcatcgag agcatgaccg gtgacgcggt gtga

[0088] SEQ ID NO.6 (Nucleotide sequence of imine reductase mutant G43V)

[0089] atgaagaccc ccgtgaccgt gctcgggctg ggtgcgatgg gacgtgcgct ggtcaccgcgctgctcgacg cggggcggtc ggtcaccgtg tggaaccgga cgcccgggaa ggcggacgaa ctggcgggcgcggtcgaggc cgcgagcgtc cacgacgcgg tgaccgcggg cgggatcgtg gtcgtgtgcc tctacgaccacgcctcggtg cacgagacac tcgacccggt ggccgccggg ctgcgcggac gggcggtggt gaacctcacgaccacgacgc ccgccgaggc acgggagctc gcggagtggg cggacggcca cggaatcgag tacctcgacggagcgatcat ggcgacgccg ccgatgatcg gtgctccggg cgcgcagatc ctctacagcg ggtcgcgtgcggtgttcgat gggcatcgcg aggcgctcga cctgtgggcg acgtccgtct acgacggtgc ggacgccggaatggcgtcgc tgttcgacct cgcgatcctt tcgggcatgt atccgctgtt cgccggattc ctgcacggcgccgcgatggt ccgcgccgag ggcgtcccgg cggccgagtt cgcggagcgc gccgcgcctt tcctcgccgcgatgaccggg tcgttcgccg gcatcgcgcg aaccgtggac ggcggcggct acgacgtgcc ggggcagagcctcgactgga cggccggcgc gctggaggcc atcggccgtg cgagccgcga gcagggcgtc gatccggttccggtcgacat ggtgggggcg ctcgtccggc ggcagatcga cgaagggtac gggagcgaag atatgtcccggctcatcgag agcatgaccg gtgacgcggt gtga

[0090] SEQ ID NO.7 (Nucleotide sequence of imine reductase mutant L89V)

[0091] atgaagaccc ccgtgaccgt gctcgggctg ggtgcgatgg gacgtgcgct ggtcaccgcgctgctcgacg cggggcggcc ggtcaccgtg tggaaccgga cgcccgggaa ggcggacgaa ctggcgggcgcggtcgaggc cgcgagcgtc cacgacgcgg tgaccgcggg cgggatcgtg gtcgtgtgcc tctacgtccacgcctcggtg cacgagacac tcgacccggt ggccgccggg ctgcgcggac gggcggtggt gaacctcacgaccacgacgc ccgccgaggc acgggagctc gcggagtggg cggacggcca cggaatcgag tacctcgacggagcgatcat ggcgacgccg ccgatgatcg gtgctccggg cgcgcagatc ctctacagcg ggtcgcgtgcggtgttcgat gggcatcgcg aggcgctcga cctgtgggcg acgtccgtct acgacggtgc ggacgccggaatggcgtcgc tgttcgacct cgcgatcctt tcgggcatgt atccgctgtt cgccggattc ctgcacggcgccgcgatggt ccgcgccgag ggcgtcccgg cggccgagtt cgcggagcgc gccgcgcctt tcctcgccgcgatgaccggg tcgttcgccg gcatcgcgcg aaccgtggac ggcggcggct acgacgtgcc ggggcagagcctcgactgga cggccggcgc gctggaggcc atcggccgtg cgagccgcga gcagggcgtc gatccggttccggtcgacat ggtgggggcg ctcgtccggc ggcagatcga cgaagggtac gggagcgaag atatgtcccggctcatcgag agcatgaccg gtgacgcggt gtga

[0092] SEQ ID NO.8 (Nucleotide sequence of imine reductase mutant G43V-L89V)

[0093] atgaagaccc ccgtgaccgt gctcgggctg ggtgcgatgg gacgtgcgct ggtcaccgcgctgctcgacg cggggcggtc ggtcaccgtg tggaaccgga cgcccgggaa ggcggacgaa ctggcgggcgcggtcgaggc cgcgagcgtc cacgacgcgg tgaccgcggg cgggatcgtg gtcgtgtgcc tctacgtccacgcctcggtg cacgagacac tcgacccggt ggccgccggg ctgcgcggac gggcggtggt gaacctcacgaccacgacgc ccgccgaggc acgggagctc gcggagtggg cggacggcca cggaatcgag tacctcgacggagcgatcat ggcgacgccg ccgatgatcg gtgctccggg cgcgcagatc ctctacagcg ggtcgcgtgcggtgttcgat gggcatcgcg aggcgctcga cctgtgggcg acgtccgtct acgacggtgc ggacgccggaatggcgtcgc tgttcgacct cgcgatcctt tcgggcatgt atccgctgtt cgccggattc ctgcacggcgccgcgatggt ccgcgccgag ggcgtcccgg cggccgagtt cgcggagcgc gccgcgcctt tcctcgccgcgatgaccggg tcgttcgccg gcatcgcgcg aaccgtggac ggcggcggct acgacgtgcc ggggcagagcctcgactgga cggccggcgc gctggaggcc atcggccgtg cgagccgcga gcagggcgtc gatccggttccggtcgacat ggtgggggcg ctcgtccggc ggcagatcga cgaagggtac gggagcgaag atatgtcccggctcatcgag agcatgaccg gtgacgcggt gtga MKTPPVTAVSGLVMGTVAGTPALSDAGGSGTWNGPGKAGTNAGAGSEAEVHDAGTGGSIVVVCLYVHTVHAETSDPVAAGLRGDGGLNLTTDAPEAEGELAEVGDAHGIEYLDEADHGDDIGLPGRQILYSGAVAVFDGIAEAELDWADVRTTAVDAGNGVAFDLRIPFGMYPCFRILHGARMGPAEGVPGAEFGERARLPFLPAMTGVPAGAVFGERGRYDPGQSPDWTAARLEAIGPVRAEQGVIRFQVDMGGLVRAGIDEGYGESKYVPASIESTMTVTV

[0094] SEQ ID No.9 (Amino acid sequence of commercial glucose dehydrogenase for reaction)

[0095] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0096] SEQ ID NO.10 (commercial glucose dehydrogenase nucleotide sequence for reaction)

[0097] atgtatccgg atctgaaagg caaggtggtg gccattaccg gtgccgcagag tggtctgggtaaagcgatgg ccatccgctt cggcaaggag caagccaagg tggtgatcaa ctactacagc aacaagcaagatccgaacga agtgaaagaa gaagtgatca aagccggtgg tgaagcggtt gtggtcaag gcgacgtgaccaaagaa gacgtgaaga acatcgtgca gacggcgatc aaggagttcg gcacgctgga catcatgatcaacaacgccg gtctggaaaa cccagtgcca agccacgaga tgccgctgaa ggattgggat aaggtgatcggcacaatct gacggcgcg tttctggta gccgtgaggc gatcaagtac ttcgtggaga acgacatcaagggcaacgtg attaacatga gcagcgtgca cgaggtgatc ccgtggccgc tgttcgtgca ttacgccgcagagcaagggcg gcattaact catgaccgaa acgctggcgc tggaatatgc cccgaaaggc atccgcgtgaataacatcgg cccgggcgcc atcaacaccc cgatcaatgc ggagaagttc gccgatccga aacagaaggccgacgtggaa agcatgatcc caatgggcta catcggcgaa ccagaggaa ttgcggccgt tgccgcgtggctggccagca aagaagcgag ctacgttacc ggtatcacgc tgtttgccga tggcggcatg acgcagtatccgagtttcca agccggtcgc ggctaa

[0098] It should be understood by those skilled in the art that, although specific embodiments of the present invention are described herein for the purpose of illustration, various modifications may be made thereto without departing from the spirit and scope of the present invention. Therefore, the specific embodiments and examples of the present invention should not be construed as limiting the scope of the present invention. The present invention is only limited by the appended claims. All documents cited in this application are fully incorporated herein by reference.

Claims

1. An imine reductase mutant, wherein the amino acid sequence of the imine reductase mutant is an amino acid sequence mutated on the amino acid sequence shown in SEQ ID NO.1, and the mutation site is one of the following sites: the 44th G mutates to V or the 89th L mutates to V or the 44th G and the 89th L mutate to V at the same time.

2. The imine reductase mutant according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. The imine reductase mutant according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

4. The imine reductase mutant according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

5. A recombinant plasmid, characterized in that: A gene encoding the imine reductase mutant according to claim 1.

6. A host cell, characterized in that Contains the recombinant plasmid according to claim 5.

7. Use of the imine reductase mutant according to claim 1 in the synthesis of the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine.

8. A method for using an imine reductase mutant in the synthesis of an intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine, comprising combining the imine reductase mutant described in claim 1 with glucose dehydrogenase, using β-nicotinamide adenine dinucleotide disodium salt as a coenzyme and 5-(3,4-dihydro-2H-pyrrol-5-yl)-2-methylpyridine as a substrate, and performing asymmetric hydrogenation reduction to prepare the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine.

9. The application method according to claim 8, characterized in that: The pH value during the asymmetric hydrogenation reduction process for preparing the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine is 6.8-8.

0.

10. The application method according to claim 8, characterized in that: The reaction temperature during the asymmetric hydrogenation reduction process for preparing the intermediate (S)-2-methyl-5-(pyrrol-2-yl)pyridine is 15-35°C.

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