Imine reductase mutant and use thereof

Abstract

The present invention relates to the field of biocatalytic synthesis technology and designs an improved imine reductase mutant for use in the synthesis of (S)-3-(pyrrolidin-2-yl)pyridine, a key intermediate in the synthesis of nicotine. The invention provides an improved imine reductase, namely, an imine reductase mutant having an amino acid sequence of SEQ ID NO: 5 (SEQ ID NO: 5), wherein the optional mutations include at least one of the following: an A at position 246 or a D at position 285 to a V. This imine reductase mutant exhibits higher enzymatic activity than existing technologies, improves the yield of the catalytic reduction reaction, and shortens the reaction time.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalytic synthesis technology, and in particular relates to an imine reductase mutant in the synthesis of a key chiral intermediate of nicotine ( S )-3-(pyrrolidin-2-yl)pyridine. Background Art

[0002] Nicotine, also known as nicotine, has a chemical name of 1-methyl-2-(3-pyridyl)pyrrolidine. It is a naturally occurring liquid alkaloid with important applications in the tobacco industry, fine chemicals, pharmaceuticals, organic synthesis, and agriculture. High-purity nicotine, in particular, has become a hot commodity in the international market. S )-3-(Pyrrolidin-2-yl)pyridine is a key chiral intermediate in the synthesis of nicotine. The most advantageous preparation process reported so far is the use of imine reductase to catalyze the preparation of 3-(1-pyrrolin-2-yl)pyridine, which can achieve high optical purity.

[0003] Patent WO2020098978 A1 discloses the use of imine reductase to reduce 3-(1-pyrrolin-2-yl)pyridine, specifically disclosing 9 imine reductases that can be used to reduce 3-(1-pyrrolin-2-yl)pyridine, using glucose dehydrogenase / glucose as a coenzyme regeneration system, and performing ( S )-protonicotinamide. These imine reductases include IRED-A, IRED-B, IRED-C, IRED-D, IRED-E, IRED-F, IRED-P, IRED-X, and IRED-AB. At a 3-(1-pyrrolin-2-yl)pyridine concentration of 400 mmol / L (58 g / L), IRED-C achieved a 24-hour conversion rate of 99.6% and an ee value of 99.8%. However, at a 3-(1-pyrrolin-2-yl)pyridine concentration of 1 mol / L (146 g / L), IRED-C achieved a 24-hour conversion rate of only 52.4% and an ee value of 99.6%. Its poor catalytic activity towards 3-(1-pyrrolin-2-yl)pyridine makes it difficult to scale up production and results in high production costs. Finding imine reductases from different biological sources and modifying the enzyme molecules with protein engineering methods to obtain more efficient imine reductases is the key to achieving the chiral intermediate of nicotine ( S )-3-(pyrrolidin-2-yl)pyridine enzymatic industrial production. Summary of the Invention

[0004] Purpose of the invention: To prepare ( S The invention overcomes the deficiency of )-3-(pyrrolidin-2-yl)pyridine, provides a preparation process suitable for industrial production, and provides the society with high-quality and low-cost raw materials.

[0005] Technical solution: The present invention is based on Nocardiopsis alba The improved imine reductase and its encoding gene based on the natural imine reductase of the strain are used to catalyze the synthesis of precursor 3-(1-pyrrolidine-2-yl)pyridine using the imine reductase mutant. S )-3-(pyrrolidin-2-yl)pyridine, the reaction formula is as follows:

[0006]

[0007] In the formula: NAD is a biological term, its Chinese name is nicotinamide adenine dinucleotide, abbreviated as coenzyme; (P) indicates that the P in the bracket is optional, and when it is present, it is NADP.

[0008] NADP is the abbreviation of nicotinamide adenine dinucleotide phosphate, formerly known as triphosphate pyridine nucleotide (TPN) or coenzyme dehydrogenase II or oxidized coenzyme II.

[0009] The present invention provides an improved imine reductase, namely an imine reductase mutant, wherein the enzymatic activity and stereoselectivity of the imine reductase mutant are higher than those of the wild-type imine reductase and IRED-C in patent WO2020098978 A1.

[0010] The technical solution of the present invention is an imine reductase mutant, whose amino acid sequence is a mutant of the amino acid sequence shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 5), and the optional mutation sites include at least one of the following sites: A at position 246 or D at position 285 is mutated to V.

[0011] The amino acid sequence of the imine reductase mutant of the present invention is shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

[0012] According to another aspect of the present invention, a recombinant plasmid is provided, which contains the nucleotide sequence of any one of the above genes. Further, the plasmid is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+ ), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C, etc.

[0013] According to another aspect of the present invention, a host cell is provided, which contains any of the above-mentioned recombinant plasmids, and the host cell includes a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is preferably an Escherichia coli BL21 (DE3) cell.

[0014] According to another aspect of the present invention, there is also provided the use of an imine reductase mutant in the preparation of (S)-3-(pyrrolidin-2-yl)pyridine, comprising: in the presence of the imine reductase mutant, using 3-(1-pyrrolin-2-yl)pyridine as a substrate, subjecting the obtained product to asymmetric catalytic hydrogenation to obtain (S)-3-(pyrrolidin-2-yl)pyridine.

[0015] Specifically, in the use of the above-mentioned imine reductase mutant in the preparation of (S)-3-(pyrrolidin-2-yl)pyridine, the catalytic hydrogenation reaction is controlled with a phosphate buffer solution at a pH range of 6.8-7.8, preferably 7.0-7.2, and sodium hydroxide is used to adjust the pH during the reaction.

[0016] During the reaction, when the pH is lower than 6.8 or higher than pH 8.0, the enzyme-catalyzed reaction rate and yield are significantly reduced.

[0017] Specifically, in the use of the above-mentioned imine reductase mutant in the preparation of (S)-3-(pyrrolidin-2-yl)pyridine, the catalytic hydrogenation reaction temperature is controlled at 20-35° C., preferably 25-30° C. If the reaction temperature is lower than 20° C. or higher than 40° C., the enzyme-catalyzed reaction rate will be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Imine reductase and its mutants catalyze the asymmetric reduction of 3-(1-pyrrolin-2-yl)pyridine;

[0019] Figure 2 . Protein electrophoresis detection results of imine reductase mutants in preferred embodiment 6 of the present invention: wherein 1 represents the wild-type imine reductase parent; 2 represents the A246V mutant; 3 represents the D285V mutant; 4 represents the A246V-D285V mutant.

[0020] Beneficial effects: The technical solution of the present invention uses a random mutagenesis molecular biology method to mutate the gene of the imine reductase shown in SEQ ID NO: 1 (the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO: 5), thereby changing the amino acid sequence of the enzyme and achieving changes in the enzyme structure and function. Then, through a targeted screening method, an imine reductase having a mutation in at least one of the above-mentioned sites is obtained. The mutant imine reductase was used to catalyze the synthesis of (S)-3-(pyrrolidin-2-yl)pyridine from 3-(1-pyrrolin-2-yl)pyridine. Compared with the wild-type imine reductase parent, the enzyme activity was increased by about 12 times (Example 5), and it had very high stereoselectivity and conversion rate. When 100 g / L 3-(1-pyrrolin-2-yl)pyridine and 0.3 wt % crude imine reductase solution (calculated based on the wet weight of cells) were reacted for 4 h, the substrate conversion rate could reach over 99.5%, and the product ee value reached 99.3%. Compared with the currently disclosed optimal enzyme catalysis process (patent WO2020098978), the ee value of the product was 99.3%. When the concentration of A13-(1-pyrrolidine-2-yl)pyridine is 58.4 g / L, the conversion rate reaches 99.6% in 24 hours and the ee value is 99.8%. The present invention provides a technical solution with a higher substrate concentration (1.7 times) and a higher substrate conversion rate (reaction time of 4 hours compared with reaction time of 24 hours), which can further reduce ( S The industrial production cost of )-3-(pyrrolidin-2-yl)pyridine and nicotine is low, and it has good industrial application value.

[0021] Specific implementation: The present invention is further described below in conjunction with specific implementation examples, but the protection scope of the present invention is not limited thereto:

[0022] Example 1: Obtaining Nocardiopsis alba wild-type imine reductase parental recombinant plasmid

[0023] Obtained through NCBI GenBank nucleic acid database Nocardiopsis alba The imine reductase parent gene (GenBank: AFR08535.1) of the strain was codon-optimized and the full-length gene was artificially synthesized by a service provider into the pET21a(+) expression plasmid. The gene was then transformed into competent E. coli BL21(DE3) cells, plated on LB agar plates containing 100 mg / L ampicillin, and cultured overnight at 37°C. Several single colonies were selected and transferred to LB medium (containing 100 mg / L ampicillin). After culture at 37°C overnight, the recombinant plasmid was extracted using a plasmid extraction kit and verified by PCR and sequencing to obtain the recombinant plasmid encoding the imine reductase parent gene.

[0024] Example 2: Random mutagenesis of the wild-type imine reductase parent gene

[0025] According to the content described in Example 1, Nocardiopsis alba The recombinant plasmid containing the imine reductase parental gene of the strain was used as a template, and the Nocardiopsis alba Primer 5.0 was used to design and synthesize primers at both ends of the imine reductase encoding gene of the strain (Table 1). Error-prone PCR was used (materials and concentrations are shown in Table 2, reaction conditions are shown in Table 3) to obtain a linear gene fragment containing a large number of base mutations. These PCR products and the pET21a(+) expression plasmid were digested with enzymes, recovered by gel extraction, ligated, and transformed into competent Escherichia coli BL21(DE3) cells. The cells were then plated on LB agar plates containing 100 mg / L ampicillin.

[0026] Incubate overnight at 37°C. See Tables 1-3 for details.

[0027] Table 1 Random mutagenesis primer sequences

[0028]

[0029] Table 2 50 μL error-prone PCR material system

[0030]

[0031] Table 3 Error-prone PCR reaction conditions:

[0032]

[0033] Example 3: Cloning and expression of imine reductase mutants

[0034] In order to facilitate the cloning, expression and identification of imine reductase mutants, compatible restriction endonuclease sites were designed at the 5' and 3' ends of the gene. Nde I and Xho I The target gene and pET21a(+) (other expression plasmids capable of expressing proteins in E. coli can also be used) are simultaneously digested with restriction endonucleases and the DNA is recovered from the gel. The recovered target gene and larger plasmid fragments are ligated with T4 DNA ligase. The ligation product is transformed into E. coli BL21(DE3) competent cells, which are then plated on LB agar plates containing 100 mg / L ampicillin and cultured overnight at 37°C.

[0035] Pick a single colony grown on the above culture dish and inoculate it into LB liquid medium containing 100 mg / L ampicillin. Cultivate it with shaking at 37°C overnight. Collect the bacteria for plasmid extraction, PCR identification and double enzyme digestion identification. The correct recombinant plasmid is named pET21a(+)-AN, and the E. coli containing the correct recombinant plasmid is subsequently induced for expression. The above bacterial liquid is transferred to 500 mL of LB liquid medium containing 100 mg / L ampicillin and cultured with shaking at 37°C until the OD reaches 0. 600 =0.6-0.8, add IPTG to a final concentration of 0.05-0.5 mM, and induce expression at 22-25°C for 12-16 h. Then, remove the bacterial solution, centrifuge at 6000 × g for 20 min, and collect the cells. Then, freeze at -20°C for later use.

[0036] Example 4: Primary screening of imine reductase mutants

[0037] According to the procedures described in Examples 2 and 3, single colonies from the LB agar medium were picked and inoculated into 96-well plates. 1 mL of LB medium containing 100 mg / L ampicillin was pre-inoculated into each well. After shaking and incubation at 37°C, 220 rpm for 3 h, a certain amount of the inducer, isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.1 mM), was added and cultured at 25°C, 220 rpm for 16 h. The cells were harvested by centrifugation at 6000 × g for 20 min. The supernatant was discarded and the cells were resuspended in the same volume of 100 mM phosphate buffer (pH 7.0). The cells were sonicated using a high-throughput ultrasonic cell disruptor manufactured by Ningbo Xinzhi Biotechnology Co., Ltd. (40% power, 3 s on, 3 s off, total duration 10 min). The supernatant, obtained by centrifugation at 6000 × g for 20 min at 4°C, was used to obtain the crude enzyme solution of the imine reductase mutants and was initially screened for activity using a microplate reader. Add 20 μL of 2 mmol / L reduced nicotinamide adenine dinucleotide (NADH) solution, 140 μL of phosphate buffer (100 mM, pH = 7.0), 20 μL of enzyme solution, and 20 μL of 20 mmol / L 3-(1-pyrrolidine-2-yl)pyridine solution to a 96-well plate. Within 5 minutes of detection, the A 340 changes.

[0038] Enzyme activity calculation formula: Enzyme activity (U / mL) = (△A×V1×10 3 ) / (6220×t×V2)

[0039] △A: change in absorbance within t;

[0040] V1: total volume of the reaction system, mL;

[0041] V2: volume of enzyme solution added, mL;

[0042] 6220: molar extinction coefficient, L / mol / cm;

[0043] t: detection time.

[0044] Example 5: Rescreening of Imine Reductase Mutants

[0045] (1) Preparation of imine reductase mutant enzyme solution

[0046] The mutant strains with higher enzyme activity than the parent strain in Example 4 were inoculated at a 0.1% inoculum into 500 mL of LB medium containing 100 mg / L ampicillin. The culture was shaken at 37°C and 220 rpm for 5-6 h. A certain amount of the inducer, isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.1 mM), was added and induced at 25°C and 220 rpm for 16 h. The cells were then collected by centrifugation at 6000 × g. The cells were resuspended in 100 mM phosphate buffer (pH 7.0) and disrupted with an ultrasonic disruptor. The supernatant, which was the crude imine reductase mutant enzyme solution, was obtained by centrifugation at 6000 × g at 4°C for 20 min.

[0047] (2) Preparation of 3-(1-pyrrolidine-2-yl)pyridine catalyzed by imine reductase S )-3-(pyrrolidin-2-yl)pyridine

[0048] Add 0.1 g of the main raw material 3-(1-pyrrolidine-2-yl)pyridine to a 10 mL reaction bottle, add 2 mL of phosphate buffer (100 mM, pH = 7.0), add 2 mg of NADP + , 0.5 mL of the crude enzyme solution of the auxiliary enzyme (GDH, glucose dehydrogenase) and an appropriate amount of the crude enzyme solution of the above mutant were added, the pH of the reaction solution was controlled to 7.0, and the reaction was carried out at 30°C for 6 h. The conversion rate and ee value were analyzed by HPLC.

[0049] (3) Determination of the catalytic conversion rate of imine reductase

[0050] The reaction system described in (2) was treated with methanol (reaction system: methanol = 1:9), membrane filtered, and directly analyzed by HPLC. The HPLC conditions were:

[0051] Instrument: Thermo U3000 system HPLC

[0052] Column: Agilent poroshell 120 C18, 4.6 mm × 150 mm, 4 μm

[0053] Mobile phase: Use 0.01 mol / L potassium dihydrogen phosphate (adjust pH 7.8 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B. Perform linear gradient elution as shown in the table below;

[0054]

[0055] Detection wavelength: 260 nm;

[0056] Flow rate: 1.0 mL / min;

[0057] Column temperature: 40°C;

[0058] Injection volume: 10 μL.

[0059] Conversion rate calculation formula:

[0060]

[0061] Where A(P) is ( S )-3-(pyrrolidin-2-yl)pyridine peak area;

[0062] A(S) is the peak area of ​​the starting material 3-(1-pyrrolin-2-yl)pyridine.

[0063] (4) Optical purity identification of imine reductase catalytic products

[0064] The reaction system described in (2) was treated with mobile phase (reaction system: mobile phase = 1:50), membrane filtered and directly analyzed by HPLC. The HPLC conditions were:

[0065] Instrument: Thermo U3000 system HPLC

[0066] Column: Chiralpak AD-H, 4.6 mm × 250 mm, 5 μm

[0067] Mobile phase: n-hexane: ethanol: diethylamine = 90:10:0.1

[0068] Detection wavelength: 260 nm;

[0069] Flow rate: 1.0 mL / min;

[0070] Column temperature: 25°C;

[0071] Injection volume: 20 μL.

[0072] Calculation formula for optical purity of S-type product:

[0073]

[0074] Where A(S) is the target product ( S )-3-(pyrrolidin-2-yl)pyridine peak area;

[0075] A(R) is the enantiomer ( R )-3-(pyrrolidin-2-yl)pyridine peak area.

[0076] Mutants with better catalytic activity than the parent were selected for sequencing, the mutation sites were analyzed, and the catalytic activity was retested to determine that the catalytic activity and stereoselectivity of the mutants A246V (SEQ ID NO: 2, the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 6), D285V (SEQ ID NO: 3, the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 7), and A246V-D285V (SEQ ID NO: 4, the nucleotide sequence of the corresponding encoding gene is SEQ ID NO: 8) were significantly improved compared with the parent of this scheme. The results of the rescreening reaction are shown in Table 4.

[0077]

[0078] Note: Table 4 * Refers to the wet weight of each imine reductase recombinant cell required to transform 1 g of substrate. 1wt refers to the wet weight of each imine reductase mutant recombinant cell required to transform 1 g of main raw material.

[0079] The results in Table 4 illustrate that the A246V-D285V mutant is the optimal imine reductase mutant. Its catalytic efficiency for 3-(1-pyrrolin-2-yl)pyridine is about 12 times that of the wild-type imine reductase parent, and the product has a higher ee value.

[0080] Example 6: Preparation of Imine Reductase Mutant Enzyme Solution

[0081] The mutant strains from Example 5 were inoculated at a 0.01% inoculum into LB medium (500 mL / bottle, 10 bottles) containing 100 mg / L ampicillin. The cultures were shaken at 37°C, 220 rpm, and incubated for 5–6 h. A certain amount of the inducer, isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.1 mM), was added and induced at 25°C, 220 rpm, for 16 h. The cells were then harvested by centrifugation at 6000 × g. The resulting 30–40 g of cells were resuspended in 150 mL of 100 mM phosphate buffer (pH 7.0) and disrupted using a high-pressure homogenizer. The supernatant, representing the imine reductase mutant enzyme solution, was then centrifuged at 6000 × g at 4°C for 20 min.

[0082] Example 7: The imine reductase mutant shown in SEQ ID NO: 4 is ( SApplication of )-3-(pyrrolidin-2-yl)pyridine in the preparation

[0083] In a 1000 mL reaction flask, 100 g of the main raw material, 3-(1-pyrrolidine-2-yl)pyridine, 480 mL of phosphate buffer (100 mM, pH 7.0), 150 mL of crude imine reductase enzyme solution, 150 mL of crude glucose dehydrogenase enzyme solution, 204 g of glucose monohydrate, and 157 mg of oxidized coenzyme II disodium (NADP-Na2) were added. The reaction was allowed to proceed at 25 ± 3°C for 4 h. During the reaction, the pH was adjusted to 7.2 with 2 mol / L NaOH solution. The conversion rate of the reaction was 99.9% after 4 h. The reaction system was adjusted to pH 3.0 ± 0.5 with dilute hydrochloric acid, and 30 ± 5 g of activated carbon was added and heat-treated at 70 ± 5°C for 30–60 min. 50 g of diatomaceous earth was added and the system was filtered to obtain ( S )-3-(pyrrolidin-2-yl) aqueous solution, the external standard method was used to determine ( S )-3-(pyrrolidin-2-yl) was 91.5 g, the yield was 90%, and the product ee value was 99.3%.

[0084] The results showed that the imine reductase mutant represented by SEQ ID NO:4 could achieve a conversion rate of over 99.9% and a product ee value of 99.3% in the enzymatic preparation of (S)-3-(pyrrolidin-2-yl)pyridine in a reaction system of 100 g / L substrate and 0.3 wt % crude enzyme solution (calculated based on the wet weight of cells) after a reaction of 4 h. These imine reductase mutants exhibited extremely high stereoselectivity and efficiency in the enzymatic preparation of (S)-3-(pyrrolidin-2-yl)pyridine.

[0085] Comparative Example 1: The imine reductase mutant shown in SEQ ID NO: 4 is ( S Application of )-3-(pyrrolidin-2-yl)pyridine in the preparation

[0086] In a 1000 mL reaction flask, 100 g of the main raw material, 3-(1-pyrrolidine-2-yl)pyridine, 480 mL of phosphate buffer (100 mM, pH 7.0), 150 mL of crude imine reductase enzyme solution, 150 mL of crude glucose dehydrogenase enzyme solution, 204 g of glucose monohydrate, and 157 mg of oxidized coenzyme II disodium (NADP-Na2) were added. The reaction was allowed to proceed at 25 ± 3°C for 4 h. During the reaction, the pH was adjusted to 8.5 using 2 mol / L NaOH solution. The conversion rate was 23.4% after 4 h and 35.5% after 24 h. The reaction system was adjusted to pH 3.0 ± 0.5 using dilute hydrochloric acid. 30 ± 5 g of activated carbon was added and heat-treated at 70 ± 5°C for 30–60 min. 50 g of diatomaceous earth was added and the system was filtered to obtain ( S)-3-(pyrrolidin-2-yl) aqueous solution, the external standard method determined that (S)-3-(pyrrolidin-2-yl) was 36.2 g, the yield was 35.7%, and the product ee value was 99.2.

[0087] The results show that when the catalytic reaction pH is 8.5, although the product ee value is not affected, it will affect the enzyme catalytic reaction rate and the product yield.

[0088] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An imine reductase mutant, characterized in 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 mutation site is the following site: D at position 285 is mutated to V.

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

3. The recombinant plasmid according to claim 2, characterized in that The plasmids are pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+ ), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C.

4. A host cell, characterized in that Contains the recombinant plasmid according to claim 2.

5. The host cell according to claim 4, characterized in that The host cell is selected from a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is an Escherichia coli BL21 (DE3) cell.

6. The imine reductase mutant according to claim 1 is useful in converting 3-(1-pyrrolidine-2-yl)pyridine into ( S )-3-(pyrrolidin-2-yl)pyridine.

7. The use according to claim 6, characterized in that The pH of the catalytic hydrogenation reaction solution is in the range of 6.8-7.8.

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