CN109355266B

CN109355266B - Imine reductase mutant and application thereof in synthesis of optically active 1-substituted-tetrahydroisoquinoline derivative

Info

Publication number: CN109355266B
Application number: CN201811313231.2A
Authority: CN
Inventors: 郑高伟; 李�昊; 许建和; 钱小龙; 潘江
Original assignee: Suzhou Baifu Enzyme Technology Co ltd; East China University of Science and Technology
Current assignee: Suzhou Baifu Enzyme Technology Co ltd; East China University of Science and Technology
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2020-11-27
Anticipated expiration: 2038-11-06
Also published as: CN109355266A

Abstract

The invention relates to an imine reductase mutant and application thereof in synthesis of optically active 1-substituted-tetrahydroisoquinoline derivatives, belonging to the technical field of biological engineering. The mutant is a derivative protein of a new amino acid sequence formed by replacing one or more amino acid residues of threonine 123, phenylalanine 178 and glycine 228 in the amino acid sequence shown in SEQ ID No.2 with other amino acid residues. The specific activity of the imine reductase mutant can reach as high as 9.4U/mg, and can catalyze the 1-substituted-3, 4-dihydroisoquinoline derivative, particularly the asymmetric reduction of the 1-substituted-3, 4-dihydroisoquinoline derivative with large steric hindrance, so as to synthesize the corresponding 1-substituted-tetrahydroisoquinoline derivative with high optical activity, and the optical purity of the product reaches more than 99 percent, thus the imine reductase mutant has good industrial application prospect.

Description

Imine reductase mutant and application thereof in synthesis of optically active 1-substituted-tetrahydroisoquinoline derivative

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an imine reductase SnIR mutant derived from bacteria Stackebrandtia nassauensis, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the imine reductase mutant gene, a preparation method of the recombinant imine reductase mutant, and application of the recombinant imine reductase mutant in catalyzing asymmetric reduction of 1-substituted-3, 4-dihydroisoquinoline derivatives to prepare optically pure 1-substituted-tetrahydroisoquinoline derivatives.

Background

The optically pure 1-substituted-tetrahydroisoquinoline and the derivative thereof are important chiral intermediates for synthesizing medicaments. For example, optically pure (S) -1-phenyl-tetrahydroisoquinoline is a chiral intermediate in the synthesis of the drug solifenacin; optically pure (S) -1- (3, 4-bisazoleOxy) benzyl-6, 7-bis methoxy-tetrahydroisoquinoline is a chiral intermediate for the synthesis of the natural product annonaceous acetogenins; the optically pure (S) -1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-tetrahydroisoquinoline is a chiral intermediate synthesized by the drug amorenol. Among them, Solifenacin (Solifenacin), trade name "weixikang", is a muscle M developed by pharmaceutical companies in japan mountain₃Receptor antagonists, widely used clinically for the treatment of overactive bladder syndrome; annona squamosa Linn (xylophine), a natural product derived from Annona squamosa Linn, has antitussive and expectorant effects; amorphophallt (Almorexant), an orexin receptor antagonist co-developed by GSK and Actelion for the treatment of insomnia.

The chemical synthesis of chiral 1-substituted-tetrahydroisoquinoline mainly utilizes rhodium or iridium and other heavy metal catalysts to carry out asymmetric hydrogenation or transhydrogenation to ensure that the 1-substituted-3, 4-dihydroisoquinoline is asymmetrically reduced to generate the corresponding optically active 1-substituted-tetrahydroisoquinoline. For example, for (S) -1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-1, 2,3, 4-tetrahydroisoquinoline, the ee values of the obtained product were 96% and 89% respectively by the methods of chemical hydrogenation and trans-hydrogenation (org. Process Res. Dev.,2013,17, 1531-1539). However, the chemical method has great difficulty in operation, harsh reaction conditions (no water and no oxygen), and the use of the heavy metal catalyst causes environmental pollution, so that the large-scale industrial application is limited. Currently, researchers have developed various biocatalytic approaches to the synthesis of optically active tetrahydroisoquinoline derivatives, including monoamine oxidase-catalyzed deracemization processes and imine reductase-catalyzed asymmetric reduction processes. British Turner et al used monoamine oxidase mutant (MAO-N D11) and borane coupling to achieve 1-phenyl-tetrahydroisoquinoline racemization, the product (S) -1-phenyl-tetrahydroisoquinoline yield and ee value respectively up to 90% and 98% (J.Am.chem.Soc.,2013,135, 10863-10869). The screening of Dianthus superbus and the like in China obtains imine reductase IR45 which can catalyze the asymmetric reduction of 1-phenyl-3, 4-dihydroisoquinoline to obtain the product (S) -1-phenyl-tetrahydroisoquinoline (ACS Catal.,2017,7, 7003) -7007) with ee of more than 99%. The enzymatic asymmetric reduction method has the advantages of high selectivity, mild reaction conditions, simple operation and the like, so the method for synthesizing the chiral amine by the asymmetric reduction of the imine catalyzed by the enzymatic method has attracted more and more attention in recent years. However, no report has been found so far for the asymmetric reduction synthesis of corresponding chiral tetrahydroisoquinoline derivatives from compounds such as 1-benzyl-or 1-phenethyl-3, 4-dihydroisoquinoline by using imine reductase.

In previous studies, the inventor clones an imine reductase SnIR from a bacterium Stackebrandtia nasauensis, and the enzyme can catalyze asymmetric hydrogenation of various 1-substituted-3, 4-dihydroisoquinolines with high stereoselectivity to prepare corresponding optical pure cyclic 3, 4-dihydroisoquinoline derivatives (org. Lett.,2017,19, 3151-. But the activity and stereoselectivity of the enzyme to the 1-substituted-3, 4-dihydroisoquinoline compound with larger para-position resistance are very low, for example, the specific activity of the 1-phenyl-3, 4-dihydroisoquinoline is only 0.013U/mg, and the ee value of the product is only 51%; the specific activity of the 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline is only 0.168U/mg, and the ee value of the product is only 11 percent. Therefore, the catalytic performance of SnIR is modified by means of protein engineering such as site-directed mutagenesis, random mutagenesis and the like, and imine reductase which has high catalytic activity and stereoselectivity to the 1-substituted-3, 4-dihydroisoquinoline substrate with large steric hindrance is developed, so that the method has very important significance for green and efficient synthesis of the 1-substituted-tetrahydroisoquinoline compound with large steric hindrance.

Disclosure of Invention

The invention aims to overcome the problems and the defects in the prior art, the wild imine reductase SnIR is subjected to molecular modification through a protein engineering technology to obtain the imine reductase mutant with obviously improved activity and stereoselectivity, and a green enzyme method way for synthesizing optically pure 1-substituted-1, 2,3, 4-tetrahydroisoquinoline by asymmetric reduction of a 1-substituted-3, 4-dihydroisoquinoline substrate with large steric hindrance is provided.

The purpose of the invention can be realized by the following technical scheme:

the invention adopts one of the technical schemes that: provides an imine reductase SnIR mutant with improved catalytic activity and stereoselectivity to large steric hindrance 1-substituted-3, 4-dihydroisoquinoline.

Carrying out mutation on imine reductase SnIR with an amino acid sequence shown as SEQ ID No.2 in a sequence table, and replacing threonine at the 123 th site with isoleucine, or respectively or simultaneously carrying out replacement on phenylalanine at the 178 th site and glycine at the 228 th site on the basis of the mutation, so as to obtain an imine reductase mutant with improved activity and stereoselectivity derived from the imine reductase SnIR.

Namely, the mutant is a derivative protein of a new amino acid sequence formed by replacing one or more amino acid residues of threonine 123, phenylalanine 178 and glycine 228 in the amino acid sequence shown in SEQ ID No.2 with other amino acid residues.

Carrying out single point mutation on amino acids around the active site of the imine reductase SnIR, and carrying out mutant SnIR_T123IIs obtained by screening from a 123 th amino acid residue site-directed saturated mutation library of the enzyme, and the catalytic activity of the enzyme on 1-p-trifluoromethyl phenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline is obviously improved. On the basis, SnIR is subjected to random mutation and site-directed saturation mutation_T123IAnd (5) carrying out continuous modification to further enhance the catalytic activity and stereoselectivity of the enzyme.

Specifically, the imine reductase mutant is obtained by mutating the amino acid sequence of wild type imine reductase SnIR under any one of the following conditions:

(1) the 123 th threonine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by isoleucine and is named as SnIR_T123I；

(2) The 123 th threonine and the 228 th glycine of the amino acid sequence shown as SEQ ID No.2 in the sequence table are replaced by isoleucine, alanine, threonine, valine, glutamic acid or arginine, which are respectively named as SnIR_T123I/G228A、SnIR_T123I/G228T、SnIR_T123I/G228V、SnIR_T123I/G228EAnd SnIR_T123I/G228R；

(3) The 123 th threonine of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by isoleucine, the 178 th phenylalanine is replaced by alanine, serine, cysteine, threonine, methionine, isoleucine, valine or leucine, and the 228 th glycine is replaced by alanine, threonine, valine, glutamic acid or arginine.

Further, the 123 th threonine, the 178 th phenylalanine, the 178 th alanine, the serine, the cysteine, the threonine, the methionine, the isoleucine, the valine or the leucine and the 228 th glycine of the amino acid sequence shown as SEQ ID No.2 in the sequence table are replaced by isoleucine and are respectively named as SnIR_{T123I/F178A/G228A}、SnIR_{T123I/F178S/G228A}、SnIR_{T123I/F178C/G228A}、SnIR_{T123I/F178T/G228A}、SnIR_{T123I/F178M/G228A}、SnIR_{T123I/F178I/G228A}、SnIR_{T123I/F178V/G228A}And SnIR_{T123I/F178L/G228A}。

The method for obtaining the mutant specifically comprises the following steps:

1. constructing a fixed-point saturated mutation library by using a mutation primer (selecting a section of base sequence of 15-20 bp respectively at the upstream and downstream of an amino acid site to be mutated) containing 123 mutation points as a template, replacing the base of the mutation points with codons of the mutated amino acid as a PCR forward primer, and performing high-throughput screening on the obtained mutation library to obtain the mutant SnIR, wherein the upstream and downstream of the mutation primer are amino acid sites to be mutated, and the base sequence of the mutation primer is a PCR reverse primer_T123I；

2. Mutant SnIR obtained by first round mutation transformation_T123IThe gene sequence is used as a template of the second round of mutation, a random mutation library is constructed by adopting an error-prone PCR strategy, and the obtained mutation gene library is subjected to high-throughput screening to obtain the mutant SnIR with improved activity_T123I/G228R(ii) a Further constructing a fixed point saturation mutation library of the 228 th amino acid residue, and screening the obtained mutation library to obtain a mutant SnIR with improved activity_T123I/G228A、SnIR_T123I/G228T、SnIR_T123I/G228VAnd SnIR_T123I/G228E；

3. By mutant SnIR_T123I/G228AThe gene sequence is used as a template, a random mutation library is constructed by adopting an error-prone PCR strategy, and the obtained mutation gene library is subjected to high-throughput screening to obtain a mutant SnIR with greatly improved catalytic activity_{T123I/F178L/G228A}(ii) a Further mutant SnIR_{T123I/F178L/G228A}The gene sequence is used as a template, a fixed point saturation mutation library of 178 th amino acid residues is constructed, the obtained mutation gene library is screened, and the mutant SnIR is obtained_{T123I/F178A/G228A}、SnIR_{T123I/F178S/G228A}、SnIR_{T123I/F178C/G228A}、SnIR_{T123I/F178T/G228A}、SnIR_{T123I/F178M/G228A}、SnIR_{T123I/F178I/G228A}And SnIR_{T123I/F178V/G228A}Wherein the mutant SnIR_{T123I/F178A/G228A}The activity of the compound is improved by more than 50 times than that of the female parent.

The PCR amplification constructed by the site-specific saturation mutation library is a conventional technology in the field, and the PCR amplification reaction system is as follows: 0.5-20 ng of template, 1. mu.l (10. mu.M) of each pair of mutation primers, and 10. mu.l of 2 XPrimestar mix, and sterile distilled water was added to 20. mu.l.

The amplification procedure of the site-directed saturation mutagenesis PCR is as follows: (1) denaturation at 98 ℃ for 10 s; (2) denaturation at 98 ℃ for 10s, (3) annealing at 55 ℃ for 15s, (4) extension at 72 ℃ for 6.5min, 25 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation of PCR product at 12 ℃.

Wherein the error-prone PCR amplification constructed by the random mutation library is a conventional technology in the field, and the PCR amplification reaction system is as follows: 0.5-20 ng of template, 1.5. mu.l (10. mu.M) of each pair of mutation primers, and 8.75. mu.l of MnCl₂(1mM), 25. mu.l of 2 XTaq mix, sterile distilled water was added to 50. mu.l.

The error-prone PCR amplification procedure was: (1) denaturation at 95 deg.C for 3 min; (2) denaturation at 95 ℃ for 30s, (3) annealing at 55 ℃ for 30s, (4) extension at 72 ℃ for 1min, 30 cycles of steps (2) - (4), finally extension at 72 ℃ for 10min, and preservation at 12 ℃.

The second technical scheme adopted by the invention is as follows: providing an isolated nucleic acid encoding the imine reductase mutant of any of claims 1 to 3.

The preparation method of the nucleic acid is a conventional preparation method in the field, and comprises the following steps: obtaining the nucleic acid molecule for coding the imine reductase mutant by a gene cloning technology, or obtaining the nucleic acid molecule for coding the imine reductase mutant by an artificial complete sequence synthesis method.

The preparation method of the nucleic acid is a conventional preparation method in the field, and the preferred preparation method comprises the following steps: and (3) performing PCR amplification on the required target gene by taking the mutant DNA sequence obtained in the first technical scheme as a template to obtain the nucleic acid molecule with point mutation.

Wherein the PCR amplification technology is a conventional technology in the field, and the PCR amplification system is as follows: 0.5-20 ng of template, 1. mu.l (10. mu.M) of each pair of amplification primers, 10. mu.l of 2 XPrimestar mix, and 20. mu.l of sterilized distilled water.

The procedure of PCR amplification is as follows: (1) denaturation at 98 ℃ for 10 s; (2) denaturation at 98 ℃ for 10s, (3) annealing at 55 ℃ for 15s, (4) extension at 72 ℃ for 6.5min, 25 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation at 12 ℃.

The third technical scheme adopted by the invention is as follows: a recombinant expression vector comprising the above nucleic acid is provided.

Wherein said recombinant expression vector is obtainable by methods conventional in the art, i.e.: the imine reductase mutant gene is constructed by connecting the nucleic acid molecule of the imine reductase mutant gene to various commercially available expression vectors. The expression vector of the present invention is preferably the plasmid pET-28a (+). The recombinant expression vector of the present invention can be prepared by the following method: the nucleic acid product obtained by PCR amplification and an expression vector pET-28a are respectively subjected to double enzyme digestion by restriction enzymes BamH I and Hind III to form complementary cohesive ends, and the cohesive ends are connected by T4DNA ligase to form a recombinant expression plasmid containing the imine reductase gene.

The fourth technical scheme adopted by the invention is as follows: provides a recombinant expression transformant containing the recombinant expression vector.

Wherein the recombinant expression transformant is preferably prepared by: the recombinant expression vector is transformed into host microorganism to obtain the recombinant expression vector. Wherein the host microorganism is preferably: coli (e. coli), more preferably e.coli BL21(DE3) or e.coli DH5 α. Coli BL21(DE3) to obtain a preferred genetically engineered strain of the invention. The plasmid transformation method can be selected from the conventional methods in the field, such as electrotransfer method, heat shock method and the like, preferably the heat shock method is selected for transformation, plasmid solution is mixed with competent cells, the mixture is heated for 90 seconds at 42 ℃, then ice bath is carried out for 3min, then recovery is carried out for 1h at 37 ℃, and LB agar medium plates containing kanamycin are coated and cultured to obtain the target recombinant expression transformant.

The fifth technical scheme adopted by the invention is as follows: provides a preparation method of an imine reductase mutant, which comprises the following steps: culturing the recombinant expression transformant, and obtaining the recombinant imine reductase mutant from the culture.

The preparation method of the recombinant imine reductase mutant preferably comprises the following steps: the recombinant Escherichia coli was inoculated into LB medium (5 g/L yeast extract, 10g/L peptone, 10g/L NaCl, pH7.0) containing kanamycin (50. mu.g/mL), cultured overnight with shaking at 37 ℃, inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium in an amount of 1% (v/v), cultured with shaking at 37 ℃ and 180rpm in a shaker, and when the absorbance OD of the culture solution is OD₆₀₀When the concentration reaches 0.6-0.8, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mM for induction, wherein the induction temperature is 16 ℃, after 24 hours of induction, centrifuging the culture solution, and washing twice by using normal saline to obtain resting cells. The obtained resting cells were suspended in potassium phosphate buffer (KPB,100mM, pH7.0), sonicated in an ice-water bath, centrifuged, and the supernatant was collected as a crude enzyme solution of the recombinant enzyme. The crude enzyme solution is analyzed by polyacrylamide gel electrophoresis, and the recombinant protein exists in a completely soluble form in cells.

The catalyst for catalyzing 1-substituted-3, 4-dihydroisoquinoline to perform asymmetric reduction reaction to form optically active chiral 1-substituted-tetrahydroisoquinoline can be the recombinant imine reductase mutant protein or a recombinant expression transformant resting cell containing the recombinant imine reductase mutant.

The invention adopts the sixth technical scheme that: provides the application of the imine reductase mutant or the recombinant escherichia coli whole cell as a catalyst for catalyzing 1-substituted-dihydroisoquinoline derivative to perform asymmetric reduction reaction to form chiral amine.

The 1-substituted-dihydroisoquinoline derivative has a structure shown in a formula I-V.

R in the formulae I, II, III, IV, V¹、R²、R³、R⁴The groups are each independently selected from hydrogen, methoxy, trifluoromethyl or halogen.

Further, the 1-substituted-dihydroisoquinoline derivative is one of the following compounds:

compound 1: formula I, wherein R¹Is hydrogen, R²Is hydrogen;

compound 2: formula I, wherein R¹Is methoxy, R²Is methoxy;

compound 3: formula II wherein R¹Is hydrogen, R²Is hydrogen, R³Is hydrogen;

compound 4: formula II wherein R¹Is methoxy, R²Is methoxy, R³Is hydrogen;

compound 5: formula II wherein R¹Is methoxy, R²Is methoxy, R³Is chlorine;

compound 6: formula III wherein R¹Is hydrogen, R²Is hydrogen, R³Is hydrogen;

compound 7: formula III wherein R¹Is methoxy, R²Is methoxy, R³Is trifluoromethyl;

compound 8: formula IV wherein R¹Is methoxy, R²Is methoxy;

compound 9: formula V, wherein R¹Is methoxy，R²Is methoxy, R³Is methoxy, R⁴Is methoxy.

In the application of the imine reductase mutant or the recombinant escherichia coli whole cell as the catalyst to catalyze the 1-substituted-dihydroisoquinoline derivative to carry out asymmetric reduction reaction to form chiral amine, the 1-substituted-dihydroisoquinoline derivative is used as a substrate, under the existence of coenzyme NADPH, the imine reductase mutant or the recombinant escherichia coli whole cell is used to catalyze the 1-substituted-dihydroisoquinoline derivative to carry out asymmetric reduction to prepare the optically active 1-substituted-tetrahydroisoquinoline derivative, and the NADPH is oxidized to generate NADP⁺。

During the reaction, the NADP is coupled with the glucose dehydrogenation reaction catalyzed by the glucose dehydrogenase⁺The enzyme method is used for reduction and regeneration to NADPH.

The conditions of the asymmetric reduction reaction according to the present invention may be selected according to the conditions conventional in such reactions in the art, and the application is preferably: the reaction is carried out in a water phase, the concentration of the substrate is 2-200 mM, the addition concentration of a cosolvent DMSO is 0-5% (w/v), the dosage of the resting cells of the imine reductase mutant is 0.1-10 kU/L, and coenzyme NADP is additionally added⁺The concentration of (A) is 0-0.5 mM, the concentration of glucose as an auxiliary substrate is 20-300 mM, the addition amount of glucose dehydrogenase is 0.1-10 kU/L, the reaction pH is 6.0-8.0, and the reaction temperature is 25-35 ℃.

The multiple imine reductase mutants are suitable for catalyzing 1-substituted-3, 4-dihydroisoquinoline compounds with large steric hindrance to reduce to prepare optically pure 1-substituted-tetrahydroisoquinoline chiral drug intermediates. Compared with the prior art, the invention has the following advantages and effects: the imine reductase mutant has higher catalytic activity and stereoselectivity, the catalytic activity on most of the 1-substituted-3, 4-dihydroisoquinoline substrates is higher than 1U/mg, the activity on 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline is as high as 9.41U/mg and is far higher than the currently reported imine reductase, and the ee value of the synthesized product is most over 99 percent. Compared with metal catalysis, the enzymatic catalytic reaction has the advantages of high optical purity of the product, mild reaction conditions, safe operation, green and environment-friendly process and the like, and has good industrial application prospect.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1 site-directed mutagenesis of amino acid 123 of imine reductase SnIR

By using

II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) protocol.

Degenerate mutation primers containing mutation points were first designed as follows:

5’-GGGTGTCATGNNKCCGGCGCCG-3’，

5’-CGGCGCCGGMNNCATGACACCC-3’。

wherein N represents A, T, C, G mixture of four bases; k represents G, T mixture of two bases; m represents A, C mixture of two bases. The template used was wild-type imine reductase SnIR recombinant plasmid pET28a-SnIR, comprising the base sequence shown in SEQ ID No. 1.

The PCR reaction system is as follows: mu.l of each template 0.5ng, forward and reverse mutation primers (10. mu.M), 10. mu.l of 2 XPrimestar mix, and sterile distilled water to make up to 20. mu.l.

The PCR amplification procedure was as follows: (1) denaturation at 98 ℃ for 10 s; (2) denaturation at 98 ℃ for 10s, (3) annealing at 55 ℃ for 15s, (4) extension at 72 ℃ for 6.5min, 25 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation of PCR product at 12 ℃.

The amplified PCR product was digested with endonuclease Dpn I at 37 ℃ for 2h, transformed into E.coli BL21(DE3) competent cells, and spread evenly on LB agar medium plates containing 50. mu.g/mL kanamycin. Culturing overnight at 37 deg.C, selecting 96 monoclonal strains to deep-well plate, culturing the primary plate containing 300 μ L LB culture medium (containing 50 μ g/mL kanamycin) per well at 37 deg.C and 220rpm overnight, sucking 50 μ L primary seed liquid, transferring to the secondary plate containing 600 μ L LB culture medium (containing 50 μ g/mL kanamycin) per well, culturing at 37 deg.C and 220 μ L kanamycinCulturing at rpm for 3h to OD₆₀₀About 1.0, adding IPTG to a final concentration of 0.2mM for induction, further culturing at 16 ℃ for 24h, centrifuging at 3,500rpm for 10min, discarding the supernatant, adding 200. mu.L of lysate (containing 750mg/L lysozyme and 12mg/L DNase) per well, shaking to suspend the cells sufficiently, incubating at 37 ℃ for 1h, centrifuging at 3,500rpm for 10min again, pipetting 50. mu.L of supernatant to a 96-well microplate, adding potassium phosphate buffer (100mM, pH7.0) containing 1mM 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline and 0.15mM NADPH, through enzyme labeling instrument detection, mutation library activity screening is carried out, mutants with obviously improved substrate 1-p-trifluoromethyl phenethyl-6, 7-dimethyl-3, 4-dihydroisoquinoline activity are obtained through screening, and sequence determination is carried out. Using DNAMAN software to carry out sequence alignment on the sequencing result, and in the optimal mutant, 123-bit amino acid is mutated into isoleucine which is named as SnIR_T123I。

Example 2 Imine reductase mutant SnIR_T123IRandom mutation of

Application of error-prone PCR (polymerase chain reaction) technology to imine reductase mutant SnIR_T123IRandom base mutation is introduced into a gene sequence, wherein the primers are as follows:

an upstream primer: 5' -GCGGATCCAACTCCAAGAAGTCTCCCGTC-3’

A downstream primer: 5' -CCCAAGCTTCTACGCCGCCT TCTTCTTGAT-3’

Wherein the template is the imine reductase mutant SnIR obtained in example 1_T123IThe recombinant plasmid pET28a-SnIR of (1)_T123I。

The PCR reaction system is as follows: template 0.5ng, a pair of mutation primers (10. mu.M) each 1.5. mu.l, 8.75. mu.l MnCl₂(1mM), 2 XTaq mix 25. mu.l, sterile distilled water was added to 50. mu.l.

The procedure for error-prone PCR amplification was as follows: (1) denaturation at 95 deg.C for 3 min; (2) denaturation at 95 ℃ for 30s, (3) annealing at 55 ℃ for 30s, (4) extension at 72 ℃ for 1min, 30 cycles of steps (2) - (4), finally extension at 72 ℃ for 10min, and preservation at 12 ℃.

After the PCR amplification product was purified, the PCR amplification product and the vector plasmid pET28a were digested simultaneously at 37 ℃ for 12 hours with restriction enzymes BamH I and Hind III, respectively, and the digested product was recoveredAfter overnight ligation at 16 ℃ with T4DNA ligase, E.coli BL21(DE3) competent cells were transformed and plated out evenly on LB agar medium plates containing 50. mu.g/mL kanamycin and cultured overnight at 37 ℃ to construct a random mutant library. Selecting single clone to deep well plate for culture, culturing the primary plate at 37 deg.C and 220rpm with LB medium (containing 50. mu.g/mL kanamycin) 300. mu.L per well overnight, transferring 50. mu.L of the primary seed solution to the secondary plate at 37 deg.C and 220rpm with LB medium (containing 50. mu.g/mL kanamycin) 600. mu.L per well, culturing at 37 deg.C and 220rpm for 3h to OD₆₀₀About 1.0, adding IPTG to a final concentration of 0.2mM for induction, further culturing at 16 ℃ for 24h, centrifuging at 3,500rpm for 10min, discarding the supernatant, adding 200. mu.L of lysate (containing 750mg/L lysozyme and 12mg/L DNase) per well, shaking to suspend the cells sufficiently, incubating at 37 ℃ for 1h, centrifuging at 3,500rpm for 10min again, pipetting 50. mu.L of supernatant to a 96-well microplate, adding potassium phosphate buffer (100mM, pH7.0) containing 1mM 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline and 0.15mM NADPH, through enzyme labeling instrument detection, mutation library activity screening is carried out, mutants with improved activity on substrate 1-p-trifluoromethyl phenethyl-6, 7-dimethyl-3, 4-dihydroisoquinoline are obtained through screening, and sequence determination is carried out. In the optimal mutant, glycine at position 228 is mutated into arginine, and the mutant is named SnIR_T123I/G228R。

Example 3 Imine reductase mutant SnIR_T123ISite-directed saturation mutagenesis of amino acid 228

Degenerate mutation primers containing mutation points were designed as follows:

5’-GTCTGGAGCTGNNKCGCAACCTCG-3’，

5’-CGAGGTTGCGMNNCAGCTCCAGAC-3’。

wherein N represents A, T, C, G mixture of four bases; k represents G, T mixture of two bases; m represents A, C mixture of two bases. The template is the imine reductase mutant SnIR obtained in example 2_T123I/G228RThe recombinant plasmid pET28a-SnIR of (1)_T123I/G228R。

The PCR reaction system is as follows: template 0.5ng, a pair of mutation primers 1. mu.l each (10. mu.M), 2 XPrimestar mix 10. mu.l, sterile distilled water to 20. mu.l.

The PCR amplification procedure was as follows: (1) denaturation at 98 ℃ for 10 s; (2) denaturation at 98 ℃ for 10s, (3) annealing at 55 ℃ for 15s, (4) extension at 72 ℃ for 6.5min, 25 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation at 12 ℃.

The amplified PCR product was digested with endonuclease Dpn I at 37 ℃ for 2h, then E.coli BL21(DE3) competent cells were transformed and spread evenly on LB agar medium plates containing 50. mu.g/mL kanamycin. Culturing at 37 deg.C overnight, selecting 96 monoclonal strains to deep-well plate, culturing at 37 deg.C overnight at 220rpm in primary plate containing LB medium 300 μ L per well, transferring 50 μ L of primary seed liquid to secondary plate containing LB medium 600 μ L per well (containing kanamycin 50 μ g/mL), culturing at 37 deg.C for 3 hr at 220rpm to OD₆₀₀When the concentration is about 1.0, adding IPTG with 0.2mM final concentration for induction, continuously culturing for 24h at 16 ℃, centrifuging for 10min at 3,500rpm, discarding the supernatant, adding 200 microliter lysate (containing 750mg/L lysozyme and 12mg/L DNase) into each hole, oscillating to fully suspend the cells, keeping the temperature for 1h at 37 ℃, centrifuging for 10min at 3,500rpm again, respectively sucking 50 microliter supernatant to a 96-hole enzyme label plate, adding potassium phosphate buffer (100mM, pH7.0) containing 1mM 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline and 0.15mM NADPH, detecting the activity of a mutant library by an enzyme reader, screening to obtain mutants with improved activity of substrate 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline, respectively mutating 228-glycine into alanine, Threonine, valine and glutamic acid, designated as SnIR respectively_T123I/G228A、SnIR_T123I/G228T、SnIR_T123I/G228VAnd SnIR_T123I/G228E。

Example 4 Imine reductase mutant SnIR_T123I/G228ARandom mutation of

Application of error-prone PCR (polymerase chain reaction) technology to imine reductase mutant SnIR_T123I/G228AThe gene sequence of (1) is introduced with random base mutation, wherein the primers used are as follows:

an upstream primer: 5' -GCGGATCCAACTCCAAGAAGTCTCCCGTC-3’

A downstream primer: 5' -CCCAAGCTTCTACGCCGCCT TCTTCTTGAT-3’

Wherein the template is the imine reductase mutant SnIR obtained in example 3_T123I/G228AThe recombinant plasmid pET28a-SnIR of (1)_T123I/G228A。

The PCR reaction system is as follows: 0.5-20 ng of template, 1.5. mu.l (10. mu.M) of each pair of mutation primers, and 8.75. mu.l of MnCl₂(1mM), 2 XTaq mix 25. mu.l, sterile distilled water was added to 50. mu.l.

After purification of the PCR amplification product, the PCR amplification product and the vector pET28a were digested simultaneously with restriction enzymes BamH I and Hind III at 37 ℃ for 12 hours, respectively, the digested products were recovered, ligated with T4DNA ligase at 16 ℃ overnight, E.coli BL21(DE3) competent cells were transformed, and uniformly plated on LB agar medium plates containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to construct a random mutant library. Selecting single clone to deep well plate for culture, culturing the primary plate at 37 deg.C and 220rpm with LB medium (containing 50. mu.g/mL kanamycin) 300. mu.L per well overnight, transferring 50. mu.L of the primary seed solution to the secondary plate at 37 deg.C and 220rpm with LB medium (containing 50. mu.g/mL kanamycin) 600. mu.L per well, culturing at 37 deg.C and 220rpm for 3h to OD₆₀₀When the concentration is about 1.0, adding IPTG with 0.2mM of final concentration for induction, continuously culturing for 24h at 16 ℃, centrifuging for 10min at 3,500rpm, discarding the supernatant, adding 200 microliter of lysate (containing 750mg/L of lysozyme and 12mg/L of DNase) into each hole, oscillating to fully suspend the cells, preserving the temperature for 1h at 37 ℃, centrifuging for 10min at 3,500rpm again, respectively sucking 50 microliter of supernatant to a 96-hole enzyme label plate, adding potassium phosphate buffer (100mM, pH7.0) containing 1mM 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline and 0.15mM NADPH, detecting the activity of a mutant library by an enzyme label analyzer, screening to obtain mutants with greatly improved activity on substrates 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline, and determining the sequence, phenylalanine 178 was mutated to leucine and named SnIR_{T123I/F178L/G228A}。

Example 5 Imine reductaseMutant SnIR_T123I/G228ASite-directed mutagenesis of amino acid 178

5’-CACCTCGACGTGNNKCTCACGACGCTG-3’，

5’-CAGCGTCGTGAGMNNCACGTCGAGGTG-3’。

wherein N represents A, T, C, G mixture of four bases; k represents G, T mixture of two bases; m represents A, C mixture of two bases.

Wherein the template is the imine reductase mutant SnIR obtained in example 4_{T123I/F178L/G228A}The recombinant plasmid pET28a-SnIR of (1)_{T123I/F178L/G228A}。

The PCR reaction system (20. mu.l) was: 0.5-20 ng of template, 1. mu.l (10. mu.M) of each pair of mutation primers, and 10. mu.l of 2 XPrimestar mix, and sterile distilled water was added to 20. mu.l.

PCR amplification procedure: (1) denaturation at 98 ℃ for 10 s; (2) denaturation at 98 ℃ for 10s, (3) annealing at 55 ℃ for 15s, (4) extension at 72 ℃ for 6.5min, 25 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation at 12 ℃.

The amplified PCR product was digested with endonuclease Dpn I at 37 ℃ for 2h, then E.coli BL21(DE3) competent cells were transformed, and LB agar medium plates containing 50. mu.g/mL kanamycin were uniformly spread. Culturing at 37 deg.C overnight, selecting 96 monoclonal strains to deep-well plate, culturing at 37 deg.C overnight at 220rpm in primary plate containing LB medium 300 μ L per well, transferring 50 μ L of primary seed liquid to secondary plate containing LB medium 600 μ L per well (containing kanamycin 50 μ g/mL), culturing at 37 deg.C for 3 hr at 220rpm to OD₆₀₀About 1.0, adding IPTG with final concentration of 0.2mM for induction, culturing at 16 deg.C for 24h, centrifuging at 3,500rpm for 10min, discarding supernatant, adding 200. mu.L lysate (containing 750mg/L lysozyme and 12mg/L DNase) into each well, shaking to suspend cells sufficiently, incubating at 37 deg.C for 1h, centrifuging at 3,500rpm for 10min again, respectively sucking 50. mu.L supernatant to 96-well microplate, adding potassium phosphate buffer (100mM, pH7.0) containing 1mM 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline and 0.15mM NADPH, and detecting the activity of mutation library by microplate readerScreening, screening to obtain mutant with improved activity to substrate 1-p-trifluoromethyl phenethyl-6, 7-double methoxy-3, 4-dihydroisoquinoline, and sequencing to obtain mutant with 178 th site phenylalanine mutated into alanine, serine, cysteine, threonine, methionine, isoleucine and valine, named pET28a-SnIR_{T123I/F178A/G228A}、pET28a-SnIR_{T123I/F178S/G228A}、pET28a-SnIR_{T123I/F178C/G228A}、pET28a-SnIR_{T123I/F178T/G228A}、pET28a-SnIR_{T123I/F178M/G228A}、pET28a-SnIR_{T123I/F178I/G228A}And pET28a-SnIR_{T123I/F178V/G228A}。

EXAMPLE 6 construction of recombinant expression vector and preparation of recombinant expression transformant

The DNA fragments of the imine reductase mutants obtained in examples 1 to 5 were digested with restriction enzymes BamH I and Hind III for 12h at 37 ℃ respectively, purified by agarose gel electrophoresis, and the fragments were recovered using an agarose gel DNA recovery kit, and ligated with plasmid pET28a digested with BamH I and Hind III by T4DNA ligase at 16 ℃ overnight to give recombinant expression plasmids.

The recombinant expression plasmid is transformed into E.coli DH5 alpha competent cells, 10 mu L of connecting liquid is taken and added into 100 mu L of competent cells, the mixture is kept still for 30min on ice after being mixed, then heat shock is carried out for 90 s at 42 ℃, ice bath is carried out for 3min, 800 mu L of LB culture medium is added, recovery is carried out for 1h at 37 ℃, an LB agar culture medium plate containing kanamycin is coated, culture is carried out at 37 ℃, positive recombinant bacteria are screened on a resistant plate containing kanamycin, single clone is selected, and colony PCR is carried out to verify positive clone. Culturing the recombinant bacteria, extracting plasmids, re-transforming into E.coli BL21(DE3) competent cells, coating the transformation solution on an LB agar medium plate containing kanamycin, and carrying out inverted culture at 37 ℃ overnight to obtain a positive recombinant transformant.

Example 7 expression, purification and enzymatic Activity assay of imine reductase SnIR mutants

The recombinant expression strain of the imine reductase mutant obtained in example 6 was inoculated into 100mL of LB liquid medium containing 50. mu.g/mL kanamycin at 37 ℃ and 180rpmCultured to OD₆₀₀When the temperature reaches 0.6-0.8 ℃, cooling to 16 ℃, adding IPTG with the final concentration of 0.2mM for induction expression, and continuing to culture for 24 hours. After completion of the culture, the cells were collected by centrifugation and washed twice with physiological saline. The obtained resting cells are suspended in KPB buffer solution (100mM, pH7.0), and are subjected to ultrasonic disruption in an ice water bath, and supernatant fluid is collected by centrifugation, so that cell disruption solution of the recombinant imine reductase mutant is obtained. Freeze drying the cell disruption solution to obtain crude enzyme powder.

The imine reductase mutant protein is purified by Ni column affinity chromatography, and the specific method is as follows:

(1) equilibrating the affinity Ni column (5 ml bed volume) with sodium phosphate buffer (20mM, pH 7.5, containing 500mM NaCl);

(2) resuspending the obtained resting cells with sodium phosphate buffer (20mM, pH 7.5, containing 500mM NaCl), sonicating, centrifuging at 10000 Xg for 45min, collecting the supernatant, passing through a Ni column at a flow rate of 1mL/min to bind the protein of interest to the Ni column;

(3) eluting the hetero-protein which has no binding ability with the Ni column by using a sodium phosphate buffer solution (20mM, pH 7.5 and 500mM NaCl) containing 0-50 mM imidazole;

(4) eluting the target protein with sodium phosphate buffer (20mM, pH 7.5, containing 500mM NaCl) containing 250mM imidazole;

(5) the collected protein samples were subjected to SDS-PAGE electrophoretic detection. Mu.l of a sample was added to 5. mu.l of 5 XSDS loading buffer, and heat-treated, the loading amount for electrophoresis was 10. mu.l, and a standard Molecular Weight Protein (Protein Molecular Weight Marker) was purchased from Thermo corporation, USA. The concentration of SDS-PAGE gel is 12%, and 90V voltage is selected for concentration, and then 120V voltage is adopted for separation. The result shows that the molecular weight of the high-purity mutant protein obtained by the purification method is about 35 kDa. The results of the activity assay of the recombinant imine reductase by the method of detecting the change in absorbance at 340nm using 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline and NADPH as substrates are shown in Table 1.

TABLE 1 Properties of SnIR and its mutants

Example 8 different SnIR mutants catalyze asymmetric reduction of various imine substrates

In 1mL KPB buffer (100mM, pH7.0), adding a final concentration of 0.1mM of each 1-substituted-3, 4-two hydrogen isoquinoline substrate and a final concentration of 0.2mM NADPH, 30 degrees C temperature after 2 minutes, adding 100u L example 7 obtained mutant enzyme liquid, reaction for 24h, using equal volume of ethyl acetate extraction product, extract product after acetylation by HPLC analysis conversion and ee value, HPLC analysis method is shown in Table 2, the results are shown in Table 3.

TABLE 2 analysis of the product amines

TABLE 3 different SnIR mutants catalyze the conversion of 1-substituted-dihydroquinolines

Acetylation of the product: taking 0.2mL of ethyl acetate extract, placing the ethyl acetate extract in a 5mL test tube with a plug, volatilizing the dry solvent at normal temperature, then dripping 2 drops of acetic anhydride and 2 drops of pyridine, placing the mixture in a boiling water bath for reaction for 1 hour, slightly cooling, and adding a proper amount of ethyl acetate for dilution.

Example 9 catalysis of Imine reductase mutant for asymmetric reduction of 1-p-trifluoromethylphenethyl-6, 7-bis-methoxy-3, 4-dihydroisoquinoline

Take 10U of SnIR prepared as in example 7_{T123I/F178L/G228A}The lyophilized crude enzyme powder and the lyophilized crude enzyme powder of 100U glucose dehydrogenase were dissolved in 9.9mL of KPB buffer (100mM, pH7.0), 0.1mL of cosolvent DMSO, 0.5mmol of substrate 1-p-trifluoromethylphenethyl-6, 7-bismethoxy-3, 4-dihydroisoquinoline, 0.18g of glucose, and a final concentration of coenzyme NADP of 0.1mM were added⁺The reaction is carried out at 30 ℃ under magnetic stirring for 9 hours, and the conversion rate is higher than 99%. After the reaction is finished, NaOH is added to adjust the pH value to 13, then ethyl acetate with the same volume is used for extraction for three times, the extract liquor is combined, the extract liquor is washed by saturated saline solution, anhydrous sodium sulfate is added for drying overnight, the solvent is removed by rotary evaporation separation, 280mg of (S) -product is obtained, the separation yield is 77%, and the ee value of the product is higher than 99%.

EXAMPLE 10 gram-Scale preparation of (S) -1-phenyl-1, 2,3, 4-tetrahydroisoquinoline

Into a 10-L reaction vessel, 9.9L KPB buffer (100mM, pH7.0), 100g glucose, 20.7g substrate 1-phenyl-3, 4-dihydroisoquinoline (dissolved in 100ml DMSO), 0.79g coenzyme NADP⁺500g recombinant SnIR expressing prepared as in example 7_{T123I/F178L/G228A}Reacting the resting cells (27kU) with 10kU of crude glucose dehydrogenase powder at a constant temperature of 30 ℃ under mechanical stirring at 150rpm for 24 hours until the conversion rate reaches 98%, extracting the reaction product with ethyl acetate, adding a proper amount of anhydrous sodium sulfate, drying overnight, and performing rotary evaporation to remove the solvent to obtain 19.1g product (S) -1-phenyl-1, 2,3, 4-tetrahydroisoquinoline, the separation yield is 91%, and the optical purity is 99%.

The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> university of eastern China, Baifuan enzyme technology, Suzhou, Ltd

<120> imine reductase mutant and application thereof in synthesis of optically active 1-substituted-tetrahydroisoquinoline derivative

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gccgagccgt tggtggttga gggcgcgcgg ttggcggcca gcccgaccga ggcggtggcc 180

tccagcgacc tggtgatcct gagcctcacc gactaccagg cgatgtacga catcctgtcg 240

accgccgagt cggcgctggc gggacgcacc atcgtcaacc tgtcgtccga tgaccccgac 300

gtgacccggg aagccgcgaa gtgggccgcc aagcacggcg cgacgttcat cgccgggggt 360

gtcatgactc cggcgccgac cgtcggcacc gaggccgcgt atgtgttcta cagcggaccg 420

aagtcggcgt tcgacgcgca cgagccggta ctgcggcaca tcggcggacc ccggttcctc 480

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Met Asn Ser Lys Lys Ser Pro Val Thr Leu Ile Gly Leu Gly Pro Met

1 5 10 15

Gly Gln Ala Met Val Arg Thr Leu Leu Gly Gln Gly His Pro Val Thr

20 25 30

Val Trp Asn Arg Thr Pro Ser Arg Ala Glu Pro Leu Val Val Glu Gly

35 40 45

Ala Arg Leu Ala Ala Ser Pro Thr Glu Ala Val Ala Ser Ser Asp Leu

50 55 60

Val Ile Leu Ser Leu Thr Asp Tyr Gln Ala Met Tyr Asp Ile Leu Ser

65 70 75 80

Thr Ala Glu Ser Ala Leu Ala Gly Arg Thr Ile Val Asn Leu Ser Ser

85 90 95

Asp Asp Pro Asp Val Thr Arg Glu Ala Ala Lys Trp Ala Ala Lys His

100 105 110

Gly Ala Thr Phe Ile Ala Gly Gly Val Met Thr Pro Ala Pro Thr Val

115 120 125

Gly Thr Glu Ala Ala Tyr Val Phe Tyr Ser Gly Pro Lys Ser Ala Phe

130 135 140

Asp Ala His Glu Pro Val Leu Arg His Ile Gly Gly Pro Arg Phe Leu

145 150 155 160

Gly Glu Asp Thr Gly Leu Ala Gln Leu Tyr Tyr Leu Ala His Leu Asp

165 170 175

Val Phe Leu Thr Thr Leu Ala Ser Val Val His Ala Thr Ala Leu Val

180 185 190

Ser Ala Ala Gly Val Asp Glu Ala Ala Phe Ala Pro Glu Ala Ile Arg

195 200 205

Met Val Ile Glu Thr Gly Gln Met Leu Ala Ala Glu Ala Glu Thr Gly

210 215 220

Leu Glu Leu Gly Arg Asn Leu Ala Ser Gly Asn His Pro Gly Glu Leu

225 230 235 240

Ala Thr Ala Val Met Met Gly Ala Thr Ala Asp His Ile Val Ser Ala

245 250 255

Ala Lys Gly Ser Gly Val Asp Leu Val Leu Pro Glu Ala Val Lys Ser

260 265 270

Leu Tyr Asp Arg Thr Val Ala Ala Gly His Gly Lys Asp Ser Trp Thr

275 280 285

Ala Met Tyr Glu Ile Ile Lys Lys Lys Ala Ala

290 295

Claims

1. An imine reductase mutant, characterized by: the amino acid sequence of the imine reductase mutant is one of the following sequences:

(1) replacing threonine 123 th position of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine;

(2) replacing 123 th threonine and 228 th glycine of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine and alanine, threonine, valine, glutamic acid or arginine;

(3) the 123 th threonine of the amino acid sequence shown as SEQ ID number 2 in the sequence table is replaced by isoleucine, the 178 th phenylalanine is replaced by alanine, serine, cysteine, threonine, methionine, isoleucine, valine or leucine, and the 228 th glycine is replaced by alanine, threonine, valine, glutamic acid or arginine.

2. The imine reductase mutant according to claim 1, wherein the amino acid sequence of the imine reductase mutant is one of the following sequences:

(2) replacing threonine 123 of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine, and replacing glycine 228 with alanine;

(3) replacing 123 th threonine and 228 th glycine of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine and threonine;

(4) replacing threonine 123 of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine, and replacing glycine 228 with valine;

(5) replacing 123 th threonine and 228 th glycine of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine and glutamic acid respectively;

(6) replacing threonine 123 of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine, and replacing glycine 228 with arginine;

(7) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, alanine and alanine respectively;

(8) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, serine and alanine respectively;

(9) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, cysteine and alanine respectively;

(10) replacing 123 th threonine, 178 th phenylalanine and 228 th glycine of an amino acid sequence shown as SEQ ID number 2 in a sequence table with isoleucine, threonine and alanine respectively;

(11) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, methionine and alanine respectively;

(12) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, isoleucine and alanine respectively;

(13) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, valine and alanine respectively;

(14) the 123 th threonine, the 178 th phenylalanine and the 228 th glycine of the amino acid sequence shown as SEQ ID number 2 in the sequence table are replaced by isoleucine, leucine and alanine respectively.

3. An isolated nucleic acid, characterized in that: the nucleic acid encodes an imine reductase mutant according to claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid of claim 3.

5. A recombinant expression transformant comprising the recombinant expression vector of claim 4.

6. A method for preparing an imine reductase mutant is characterized in that: culturing the recombinant expression transformant according to claim 5 to obtain an imine reductase mutant from the culture.

7. The use of an imine reductase mutant as defined in claim 1 or 2 for catalyzing asymmetric reduction of a 1-substituted-dihydroisoquinoline derivative to synthesize an optically active 1-substituted-tetrahydroisoquinoline derivative.

8. The use of claim 7, wherein: 1-substituted-dihydroisoquinoline derivative is used as a substrate, imine reductase mutant is used for catalyzing the asymmetric reduction of the 1-substituted-dihydroisoquinoline derivative to prepare the optically active 1-substituted-tetrahydroisoquinoline derivative in the presence of coenzyme NADPH, and the NADPH is oxidized to generate NADP⁺。

9. Use according to claim 8, wherein the NADP is coupled to a glucose dehydrogenation reaction catalysed by a glucose dehydrogenase⁺The enzyme method is used for reduction and regeneration to NADPH.

10. Use according to any one of claims 7 to 9, wherein: the imine reductase mutant is used in the form of enzyme protein or recombinant expression transformant resting cells.

11. The use of claim 7, wherein: the 1-substituted-dihydroisoquinoline derivative has a structure shown in a formula I-V:

12. The use of claim 11, wherein: the 1-substituted-dihydroisoquinoline derivative is one of the following compounds: