CN115838697A - Imine reductase mutant and application thereof in synthesis of chiral intermediate of erlotinib - Google Patents

Abstract

The invention relates to an imine reductase mutant and application thereof in synthesis of a chiral intermediate of erlotinib, and belongs to the technical field of biological engineering. The specific activity of the imine reductase mutant is up to 19.2U/mg, the asymmetric reduction of 2- (2, 5-difluorophenyl) pyrroline can be efficiently catalyzed, the chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine of the larotinib is synthesized, the concentration of a substrate in an enzymatic reaction is up to 80g/L, the space-time yield of a product is up to 350g/L/d, and the optical purity is up to more than 99.5%. In addition, the imine reductase mutant also has high catalytic activity on some 2-aryl substituted pyrrolines, and the optical purity of the product is as high as more than 99%. The mutant has extremely high catalytic activity, substrate tolerance and space-time yield, and has a strong industrial application prospect in the synthesis of chiral 2-arylpyrrolidine, particularly Laretinib chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine.

Description

Imine reductase mutant and application thereof in synthesis of chiral intermediate of erlotinib

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an imine reductase mutant and 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 2- (2, 5-difluorophenyl) pyrroline and analogues thereof to preparation of Laretinib chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine and chiral 2-arylpyrrolidine.

Background

Chiral 2-arylpyrrolidine is used as an azacyclic chiral amine building block and is widely used for synthesis of drug molecules, for example, a compound LY 245639 developed by the Li Lai company is a kappa opioid receptor antagonist with high affinity and high selectivity, can block stimulation mediated by a body-derived kappa agonist or an exogenous agonist such as morphine and the like, has more than 30 times of functional selectivity relative to mu and delta opioid receptors, and can be applied to addiction treatment and anti-major depression treatment. Another compound, LY27950503, is also a (KOR) PET radiation kappa opioid receptor antagonist with similar effect. The myosin-related kinase (TRK) receptor family is involved in neuronal development, and when aberrant fusion occurs, oncogenic TRK fusions induce cancer cell proliferation and are involved in key downstream signaling pathways associated with cancer. Larostinib (LOXO-101) is the first anticancer drug targeting a solid tumor of the ATP binding site of the TRK receptor family, and the molecular structure of the drug is shown in the following, and the drug molecule is already marketed in the United states in 2018.

As for the synthesis of chiral 2-substituted pyrrolidines, helmchen performed an asymmetric hydrogenation reaction of allylamine in 2009 using a rhodium catalyst (Synlett, 2009,4, 1413-1416) to synthesize various chiral 2-substituted pyrrolidines. Zhang utilizes an Ir-f-Binaphane catalyst (Advanced Synthesis & Catalysis,2010, 352. However, these chemical processes not only use heavy metal catalysts such as iridium, rhodium, ruthenium, etc., resulting in a significant increase in removal cost, but also require complicated protection and deprotection processes during the reaction. Therefore, a more green, efficient and safe synthesis method is urgently developed.

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. For the enzymatic synthesis of 2-substituted pyrrolidines, turner topic group (chemcat chem,2015,7 579-583, chemcat chem,2013,5, 3505-3508) screened imine reductase RIR and SIR with R and S selectivity, respectively, but as the substituents become larger, the enzyme selectivity becomes lower and the catalytic activity becomes lower, failing to meet the synthesis needs for the bulky hindered substrate 2-arylpyrrolidines. In 2016, the Turner topic group (ACS Catalysis,2016, 6. Recently, some natural imine reductases (Organic Letters,2020,22, 3367-3372, org. Process res. Dev.2022,26, 2067-2074) are obtained by screening 2-aryl substituted pyrroline substrates, and a series of asymmetric reductions of 2-aryl pyrroline substrates are realized, and the problems of low activity and low substrate concentration also exist. In recent years, imine reductase has been greatly developed to catalyze asymmetric reduction reactions, but most of natural enzymes have disadvantages such as substrate inhibition and low catalytic activity, resulting in a low substrate loading amount. The reaction with high substrate concentration requires the addition of a large amount of enzyme, so that the post-treatment is complicated, the cost is increased, and the industrial application is limited.

In previous studies, the inventor clones an imine reductase ScIR from the bacterium Streptomyces clavuligerus, which can catalyze asymmetric hydrogenation of 2- (2, 5-difluorophenyl) pyrroline and various 2-arylpyrrolines with high stereoselectivity to prepare corresponding chiral intermediates (R) -2- (2, 5-difluorophenyl) pyrrolidine and chiral 2-arylpyrrolidine (org. Lett.,2017,19, 3151-3154) of larotinib. However, the enzyme has the problems of low activity, poor thermal stability and incapability of converting a high-concentration substrate, for example, the specific activity of 2- (2, 5-difluorophenyl) pyrroline is only 0.18U/mg, and the substrate concentration is only 2g/L.

Disclosure of Invention

The invention aims to overcome the problems and the defects in the prior art, and the wild-type imine reductase ScIR is subjected to molecular modification through protein engineering technologies such as site-directed mutagenesis, random mutagenesis and the like to obtain the imine reductase mutant with remarkably improved catalytic activity, thermal stability and substrate tolerance, so that the substrate loading capacity in the reaction is improved. The efficient green enzymatic synthesis of the Laretinib chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine is realized, a green efficient enzymatic synthesis process is provided for the chiral 2-arylpyrrolidine, and the application of imine reductase in industry is accelerated.

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

the invention adopts one of the technical schemes that: provided is a ScIR mutant of imine reductase which has improved catalytic activity, stability and substrate tolerance to 2- (2, 5-difluorophenyl) pyrroline.

A protein derived from a novel amino acid sequence comprising a mutation of imine reductase ScIR having an amino acid sequence shown in SEQ ID No.2, wherein one or more amino acid residues selected from valine at position 122, phenylalanine at position 177, methionine at position 169, tryptophan at position 185, serine at position 211, methionine at position 270, glycine at position 214, glutamic acid at position 45, asparagine at position 53 and phenylalanine at position 265 are replaced with another amino acid residue.

Wherein the nucleotide sequence of the gene editing imine reductase ScIR is shown in SEQ ID No. 1.

Wherein, the SEQ ID No.1 sequence is as follows:

the sequence of SEQ ID No.2 is as follows:

random mutation or single-point saturation mutation is carried out on imine reductase ScIR, activity detection is carried out by using substrate 2- (2, 5-difluorophenyl) pyrroline, and mutants with remarkably improved activity and thermal stability are screened. Specifically, the imine reductase mutant is obtained by mutating the amino acid sequence of wild-type imine reductase ScIR under any one of the following conditions:

(1) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 is replaced by cysteine;

(2) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan;

(3) The valine at position 122 of the amino acid sequence shown in SEQ ID No.2 is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan;

(3) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan;

(4) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan; glycine at position 214 is replaced with phenylalanine or tryptophan;

(5) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan; glycine at position 214 is replaced with phenylalanine or tryptophan; glutamic acid at position 45 is replaced by alanine or glycine, asparagine at position 53 is replaced by threonine or tryptophan and phenylalanine at position 265 is replaced by alanine, leucine or isoleucine.

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

the gene sequence of wild-type imine reductase ScIR from Streptomyces clavuligerus is taken as a template, an error-prone PCR strategy is adopted to construct a random mutation library, and high-throughput screening is carried out on each constructed mutation library to obtain the mutants.

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 template, 1.5 mul (10 mul) of each pair of mutation primers, and 8.75 mul MnCl ₂ (1 mM), 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 ℃ for 3min; (2) Denaturation at 95 ℃ for 30s, (3) annealing at 55 ℃ for 30s, (4) extension at 72 ℃ for 1min, 30 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation at 12 ℃.

The second technical scheme adopted by the invention is as follows: an isolated nucleic acid is provided that encodes the imine reductase mutant.

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 conventional in the art, and an alternative PCR amplification system is: 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.

An alternative procedure for PCR amplification is: (1) denaturation at 98 ℃ for 10s; (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 T4 DNA 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 (DE 3) or e.coli DH5 α. Coli BL21 (DE 3) 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, pH 7.0) 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 OD ₆₀₀ When the concentration reaches 0.6-0.8, isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mM is added for induction, the induction temperature is 25 ℃, after 24 hours of induction, the culture solution is centrifuged and washed twice by normal saline, and the resting cells are obtained. Suspending the obtained resting cells in potassium phosphate buffer (KPB, 100mM, pH 7.0), ultrasonically breaking in ice water bath, centrifuging, and collecting supernatant, i.e. crude enzyme solution of recombinase. 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 2- (2, 5-difluorophenyl) pyrroline and analogues thereof to carry out asymmetric reduction reaction to form optically active (R) -2- (2, 5-difluorophenyl) pyrrolidine and analogues thereof 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: the imine reductase mutant or the recombinant escherichia coli whole cell is provided to be used as a catalyst to catalyze the 2-arylpyrroline derivative to carry out asymmetric reduction reaction to form chiral amine.

The 2-arylpyrroline derivative is selected from one or more of A structures shown in the following structural formula 1a-1 l:

in the application of the imine reductase mutant or the recombinant escherichia coli whole cell as the catalyst to catalyze the asymmetric reduction reaction of the 2-arylpyrroline derivative to form chiral amine, the 2-arylpyrroline derivative is used as a substrate, the imine reductase mutant or the recombinant escherichia coli whole cell is used for catalyzing the asymmetric reduction of the 2-arylpyrroline derivative to prepare optically active (R) -2- (2, 5-difluorophenyl) pyrrolidine and analogues thereof in the presence of coenzyme NADPH, 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 2-80g/L, the addition concentration of 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 the glucose dehydrogenase is 0 to 0.5mM, the concentration of the glucose serving as an auxiliary substrate is 20 to 700mM, the addition amount of the glucose dehydrogenase is 0.1 to 10kU/L, the reaction pH is 6.0 to 8.0, and the reaction temperature is 20 to 40 ℃.

The multiple imine reductase mutants are suitable for catalyzing 2- (2, 5-difluorophenyl) pyrroline and analogues thereof to reduce and prepare optically pure (R) -2- (2, 5-difluorophenyl) pyrrolidine and chiral drug intermediates of the analogues thereof.

Compared with the prior art, the invention has the following advantages and effects: the imine reductase mutant has higher catalytic activity and thermal stability, the catalytic activity on a 2- (2, 5-difluorophenyl) pyrroline substrate is up to 19.2U/mg, which is far higher than that of the imine reductase reported at present, the imine reductase mutant can efficiently catalyze the asymmetric reduction of the 2- (2, 5-difluorophenyl) pyrroline to synthesize a chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine of the larotinib, the substrate concentration in an enzymatic reaction is up to 80g/L, the space-time yield of a product is up to 350g/L/d, the optical purity is up to more than 99.5%, and the substrate concentration and the space-time yield are far higher than those of the imine reductase catalyzed process reported at present. 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 random mutagenesis of the imine reductase ScIR

Random base mutations were introduced into the imine reductase ScIR gene sequence using error-prone PCR techniques with the following primers:

an upstream primer (shown as SEQ ID No. 3):

5’-AACTTTAAGAAGGAGATATACCATGGGCCATCATCATCATCATCACATGTCCCGCCCCGCGCCCCTCACC-3’

the downstream primer (shown as SEQ ID No. 4):

5’-GTGGTGGTGCTCGAGTGCGGCCGCTCAGGACGGCTTCTTCAGCAGCTCC-3’

the template is a recombinant plasmid pET28a-ScIR of imine reductase ScIR.

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 MnCl ₂ (1 mM), 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 ℃ for 3min; (2) Denaturation at 95 ℃ for 30s, (3) annealing at 55 ℃ for 30s, (4) extension at 72 ℃ for 1min, 30 cycles of steps (2) - (4), final extension at 72 ℃ for 10min, and preservation at 12 ℃.

After purification of the PCR amplification product, the PCR amplification product and the vector plasmid pET28a were subjected to double digestion with restriction enzymes BamH I and Hind III at 37 ℃ for 12 hours, respectively, the digested products were recovered, ligated with T4 DNA ligase at 16 ℃ overnight, and E.coli BL21 (DE 3) competent cells were transformed and spread uniformly 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 primary seed liquid 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 OD ₆₀₀ At about 1.0, the cells were further cultured at 16 ℃ for 24h, centrifuged at 500rpm for 10min, the supernatant was discarded, 200. Mu.L of a lysis solution (containing 750mg/L of lysozyme and 12mg/L of DNase) was added to each well, the cells were thoroughly suspended by shaking, incubated at 37 ℃ for 1h, centrifuged at 500rpm for 10min again, 50. Mu.L of the supernatant was pipetted onto a 96-well microplate, a potassium phosphate buffer (100 mM, pH 7.0) containing 1mM 2- (2, 5-difluorophenyl) pyrroline and 0.15mM NADPH was added thereto, and the mutant whose activity was improved in the substrate 2- (2, 5-difluorophenyl) pyrroline was screened by screening with a microplate reader to determine the sequence. A total of 11 random mutations were performed.

Example 2 expression, purification and enzymatic Activity assay of imine reductase ScIR mutants

Selecting a recombinant expression single colony of the imine reductase mutant growing on the plate, inoculating the recombinant expression single colony into a 4mL LB test tube containing Kan (50 ng/. Mu.L), putting the LB test tube into a 37℃ shaking table, culturing for 12-16h under the condition of 200rpm, transferring the bacterial liquid into 100mL LB culture medium containing 50 ng/. Mu.L Kan according to the inoculation ratio of 1 ₆₀₀ When the concentration reached 0.6 to 0.8, 20. Mu.L of IPTG (1M) was added to induce expression of the target protein, and after culturing at 25 ℃ and 180rpm for 24 hours, the cells were collected by centrifugation. The resulting resting cells were suspended in KPB buffer (100 mM,pH 7.0), ultrasonically crushing in an ice-water bath, centrifuging and collecting supernatant to obtain cell crushing liquid of the recombinant imine reductase mutant. 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) Equilibration of 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 target protein to the Ni column;

(3) Eluting the hetero-protein which has no binding ability with Ni column with sodium phosphate buffer solution (20mM, pH 7.5, containing 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 activity of the recombinant imine reductase was measured by a method of detecting the change in the absorbance at 340nm using 1mM of 2- (2, 5-difluorophenyl) pyrroline and 0.15mM of NADPH in potassium phosphate buffer (100mM, pH 7.0) as substrates, and the results are shown in Table 1.

TABLE 1 Properties of ScIR and its mutants

Wherein, scIR _WT Refers to a natural imine reductase ScIR with an amino acid sequence shown as SEQ ID No. 2.

Example 3 ScIR and mutants thereofScIRM ₃ Activity on various 2-substituted pyrrolines

The ScIR and the mutant ScIR thereof are detected by using various 2-substituted pyrrolines 1a-1i as a substrate for detecting activity _M3 The viability assay was performed and the results are shown in table 2. Mutant ScIR for most substrates _M3 Compared with the female parent, the vitality is greatly improved. For 1d,1g,1h and 1k, even breakthrough of viability was achieved, indicating that the mutants have better universality for 2-arylpyrroline substrates.

TABLE 2 ScIR and its mutant ScIR _M3 Activity on various 2-substituted pyrrolines

Example 4 Iminireductase mutants catalyze the asymmetric reduction of 2- (2, 5-difluorophenyl) pyrroline

5mL 100g/L imine reductase mutant ScIR _M1 、ScIR _M2 And ScIR _M3 Wet cell disruption solution (KPB7.0 buffer,100 mM), bmGDH 20mg/mL (lyophilized enzyme powder, 5U/mg), 0.15mM NADP ⁺ 2-5g/L substrate 2- (2, 5-difluorophenyl) pyrroline (1% DMSO), 1.5 times of equivalent of glucose, make up to 10mL with KPB7.0buffer solution, react the reaction mixture at 30 ℃, sample every time during the reaction, sample 100. Mu.L each time, add 20. Mu.L 10mM sodium hydroxide solution, add 500. Mu.L ethyl acetate, mix well. Centrifuging at 12000rpm for 5min, collecting the upper organic phase, adding appropriate amount of anhydrous sodium sulfate, drying for 8 hr, and detecting substrate reaction condition by gas chromatography. As shown in Table 3, the conversion rate of the wild enzyme was only 26% at a substrate loading of 5g/L, while the mutant ScIRM3 could convert all 80g/L of substrate within 5h, which fully indicates that the catalytic performance of the mutant is greatly improved compared with that of the parent and the space-time yield is up to 384g/L/d.

TABLE 3 ScIR and its mutants catalyze the conversion of the substrate 2- (2, 5-difluorophenyl) pyrroline

^a The reaction mixture(10mL)contained 2-100g/L substrate,5mL cell free extract(from 100g/L wet

Example 5 enzymatic Scale Synthesis of Laretinib chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine

In a 10-L reaction vessel, 9.9L of KPB buffer (100mM, pH 7.0), 1000g of glucose, 800g of substrate 2- (2, 5-difluorophenyl) pyrroline (dissolved in 100ml of DMSO), 0.5g of coenzyme NADP were added ⁺ 500g expression of recombinant ScIR _M3 The resting cells and 10kU of crude glucose dehydrogenase powder are reacted at the constant temperature of 30 ℃ under the mechanical stirring of 150rpm, the conversion rate reaches 98 percent after 5 hours, the product obtained by the reaction is extracted by ethyl acetate, a proper amount of anhydrous sodium sulfate is added for drying overnight, the solvent is removed by rotary evaporation, 656g of the product (R) -2- (2, 5-difluorophenyl) pyrrolidine is obtained, the separation yield is 82 percent, and the optical purity is 99 percent.

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.

Claims (10)

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

(1) The valine at the 122 th site of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by cysteine;

(2) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan;

(3) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan;

(4) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan; glycine at position 214 is replaced with phenylalanine or tryptophan;

(5) The valine at position 122 of the amino acid sequence shown as SEQ ID No.2 in the sequence table is replaced by cysteine, and the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; methionine 169 to phenylalanine or tryptophan, tryptophan 185 to leucine or isoleucine, serine 211 to alanine, glycine or valine, methionine 270 to glutamic acid, arginine or tryptophan; glycine at position 214 is replaced with phenylalanine or tryptophan; glutamic acid at position 45 is replaced by alanine or glycine, asparagine at position 53 is replaced by threonine or tryptophan and phenylalanine at position 265 is replaced by alanine, leucine or isoleucine.

2. An isolated nucleic acid, characterized in that: the nucleic acid encodes the imine reductase mutant of claim 1.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.

5. A method for preparing an imine reductase mutant is characterized in that: culturing the recombinant expression transformant according to claim 4, and obtaining the imine reductase mutant from the culture.

6. Use of the imine reductase mutant of claim 1 or resting cells comprising the recombinant expression transformant of claim 4 for catalyzing asymmetric reduction of 2- (2, 5-difluorophenyl) pyrroline and analogs thereof to synthesize the chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine and chiral 2-arylpyrrolidine of Rarotinib.

7. The use of claim 6, wherein: preparing Larostinib chiral intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine and chiral 2-arylpyrrolidine by using 2- (2, 5-difluorophenyl) pyrroline and analogues thereof as substrates and catalyzing biological asymmetric reduction of 2- (2, 5-difluorophenyl) pyrroline and analogues thereof by using imine reductase mutant in the presence of coenzyme NADPH, and simultaneously oxidizing the NADPH to generate NADP ⁺ 。

8. Use according to claim 7, characterized in that the NADP is coupled to a glucose dehydrogenation reaction catalyzed by glucose dehydrogenase to convert NADP into NADP ⁺ The NADPH is regenerated by enzymatic reduction.

9. Use according to any one of claims 6 to 8, wherein: the imine reductase mutant is used in the form of enzyme protein or recombinant expression transformant resting cells.

10. The use of claim 6, wherein: the 2- (2, 5-difluorophenyl) pyrroline and the analogue thereof are one of the following compounds: