Imine reductase mutant and use thereof in preparation of tofacitinib chiral amine building block

Abstract

An imine reductase mutant and the use thereof in the preparation of a tofacitinib chiral amine building block. By means of an enzymatically coupled coenzyme regeneration system, the imine reductase mutant can efficiently catalyze racemic 1-benzyl-4-methylpiperidin-3-one as a substrate to undergo dynamic kinetic resolution and asymmetric reductive amination, so as to accurately synthesize a tofacitinib chiral amine building block (3R,4R)-1-benzyl-N-4-dimethylpiperidin-3-amine. Compared with conventional resolution methods, the technology has the significant advantages of simple operation, mild reaction conditions, environmental friendliness, high product yield, low cost, etc., and exhibits excellent application prospects in the synthesis of tofacitinib chiral amine intermediates.

Description

Imine reductase mutants and their use in preparing tofacitinib chiral amine building block TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to an imine reductase, an imine reductase mutant, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene, a recombinant imine reductase catalyst and a preparation method thereof, and an application of the imine reductase mutant or the recombinant imine reductase catalyst in preparing a tofacitinib chiral amine building block. BACKGROUND

[0002] Tofacitinib is a Janus activated kinase inhibitor containing a double chiral amine structure, which can be used for treating rheumatoid arthritis, psoriasis and preventing organ transplant immune rejection and the like. Tofacitinib was listed in China on March 10, 2017, as the first oral small-molecule drug for treating rheumatoid arthritis and the like in China. Tofacitinib has attracted widespread attention at home and abroad since its listing due to its unique targeting mechanism, treatment safety, oral convenience, high compliance and the like. In the 2020 top 200 list of drug retail sales, tofacitinib ranked 65th, with a global sales of 2.47 billion US dollars.

[0003] The molecule of tofacitinib mainly consists of three parts, i.e. a double-substituted piperidine ring, a pyrimidopyrrole double ring and a cyanoacetic ester side chain. The main challenge and cost of synthesis of the drug is the synthesis of the multi-chiral amine building block. In 2002, Pfizer reported a tofacitinib drug molecule assembly strategy (WO2002096909) for the first time. First, aromatic nucleophilic substitution reaction (S N Ar reaction) occurs between chloropyrrolopyrimidine and chiral amine building block, then the benzyl protecting group is removed by hydrogenation, and finally the cyanoacetic ester side chain is added. Among them, chloropyrrolopyrimidine is relatively inexpensive, and benzyl-protected piperidine ring is also relatively convenient, so this assembly strategy has been widely used since then. In 2007, Pfizer improved the process, using dichloropyrrolopyrimidine instead of monochloropyrrolopyrimidine to obtain higher yield, and removing the extra chlorine during hydrogenation to remove the benzyl group (WO2007012953). Since then, after years of improvement, in order to improve the yield of the product and reduce the generation of by-products, a method of protecting the chloropyrrolopyrimidine with p-toluenesulfonyl (Ts) has also been used.

[0004] In 2002, Pfizer developed a method for preparing the bifunctional amine building block of tofacitinib. The racemic mixture of four configurations was obtained from piperidone by reductive amination, followed by resolution of the racemate using recrystallization. Since piperidone is difficult to support large-scale industrial production, in 2003, Pfizer used methylpyridine as a precursor, which was benzyl-protected, reduced to alcohol by sodium borohydride, and finally oxidized to piperidone. This enabled the preparation of tofacitinib bifunctional amine building block at the kilogram level (Org Process Res Dev, 2003, 7: 2168-2172). Pfizer improved the original preparation process of tofacitinib bifunctional amine building block using heavy metal combined with chiral ligand catalytic asymmetric hydrogenation in 2004 and 2007 (Org Process Res Dev, 2005, 9: 51-56).

[0005] In recent years, the method of using transition metals combined with chiral ligands to catalyze asymmetric reductive amination or asymmetric hydrogenation has been used to prepare tofacitinib bifunctional amine building block. Chengxi Li et al. developed rhodium and chiral ligand catalyzed asymmetric hydrogenation to synthesize tofacitinib bifunctional amine building block, with a yield of 98% and an er value of 98:2, but the catalyst conversion number was low (TON < 400), and subsequent multi-step reactions were needed to obtain the final chiral amine building block product, and the process was relatively complicated. In addition, this preparation method requires high-pressure H2 of 500 psi (Angew Chem Int Ed, 2019, 58: 13573-13583). This requires additional high-pressure hydrogen equipment, further increasing production costs, and introducing safety factors in production.

[0006] In 2013, Santos developed a method for preparing tofacitinib through an intermediate of chiral alcohol, but the overall yield was only 9.5% (Tetrahedron Lett, 2013, 54: 5096-5098.). In 2018, Kamble improved the preparation process of chiral alcohol building block, but the synthesis route was long, metal catalyst and chiral ligand were used, and the final yield was less than 20% (98% ee), and there were more by-products (Synth Commun, 2018, 48: 1045-1051). Obviously, the method of introducing the chiral center of tofacitinib through chiral alcohol is not suitable for industrial production.

[0007] In summary, the current synthesis method of tofacitinib chiral amine block generally relies on the resolution of racemate. However, the method is complicated and needs to be repeatedly resolved to achieve sufficient optical purity. In addition, the racemate of the double chiral center has four configurations, and the highest theoretical yield is only 25%. In addition, the asymmetric synthesis method needs to use transition metals and chiral ligands. They are not only expensive, but also difficult to remove during subsequent product separation and purification. In addition, chemical asymmetric catalysis usually needs to be carried out in a harsh environment of high temperature and high pressure, which not only generates a large amount of by-products, but also has low optical purity and yield of the product. Therefore, it is necessary to develop a more efficient, simple and green synthesis process of tofacitinib chiral amine block. SUMMARY

[0008] In view of the problems of harsh process conditions, low product yield and insufficient optical purity in the existing chemical method for producing tofacitinib chiral amine block, the present application provides an imine reductase and a mutant thereof, a nucleic acid encoding the imine reductase and the mutant thereof, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a recombinant imine reductase catalyst and its application in the synthesis of tofacitinib chiral amine block.

[0009] The imine reductase and the mutant thereof provided by the present application have high expression, strong catalytic activity, high stability, good stereoselectivity and wide catalytic substrate spectrum.

[0010] The imine reductase or the imine reductase mutant of the present application can catalyze the dynamic kinetic resolution and asymmetric reductive amination of racemic precursor ketone substrate to synthesize a single configuration of tofacitinib chiral amine block.

[0011] The object of the present application can be achieved by the following technical solutions:

[0012] The first object of the present application is to provide an isolated imine reductase, the amino acid sequence of which is shown as SEQ ID No. 1, and the imine reductase is PocIRED.

[0013] The second object of the present application is to provide an imine reductase mutant, which is a protein corresponding to a sequence derived from the amino acid sequence shown as SEQ ID No. 1 by substituting, deleting or inserting one or more amino acid residues in the amino acid sequence, and the imine reductase mutant has advantages over the imine reductase corresponding to the amino acid sequence shown as SEQ ID No. 1.

[0014] Further, the imine reductase mutant is a derivative protein of a new amino acid sequence formed by replacing one or more of the amino acid residues at positions 239 glycine, 125 proline, 40 serine, 124 valine, 127 isoleucine, 128 leucine, 72 serine, 73 tyrosine, 76 threonine, 195 lysine, 180 glycine, 220 arginine, 47 alanine, 50 valine, 210 phenylalanine, 212 lysine, 217 tryptophan, 178 isoleucine, 224 histidine, or 236 leucine of SEQ ID No. 1 with other amino acid residues. The protein has significantly improved racemic precursor ketone reductive amination activity and stereoselectivity for the corresponding tofacitinib chiral amine building block compared to the imine reductase corresponding to the amino acid sequence shown in SEQ ID No. 1, and also has improved expression and stability.

[0015] In some embodiments of the present application, the specific amino acid sequence of the imine reductase mutant is one of the following sequences:

[0016] (1) replacing the glutamine at position 239, the proline at position 125, the serine at position 40 of the amino acid sequence shown in SEQ ID No. 1 with glycine, threonine, and glutamic acid, respectively;

[0017] (2) replacing the serine at position 72, the tyrosine at position 73, the threonine at position 76, the lysine at position 195, and the glycine at position 180 of the amino acid sequence shown in SEQ ID No. 1 with asparagine, glutamine, valine, alanine, and serine, respectively;

[0018] (3) replacing the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, and the threonine at position 76 of the amino acid sequence shown in SEQ ID No. 1 with threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, and valine, respectively;

[0019] (4) Replace the glutamine at position 239 of the amino acid sequence shown in SEQ ID No.1 with glycine, the proline at position 125 with threonine, the serine at position 40 with glutamic acid, the valine at position 124 with threonine, the isoleucine at position 127 with glutamic acid, the leucine at position 128 with tyrosine, the serine at position 72 with asparagine, the tyrosine at position 73 with glutamine, the threonine at position 76 with valine, the lysine at position 195 with alanine, the glycine at position 180 with serine, and the arginine at position 220 with valine;

[0020] (5) Replace the proline at position 125 of the amino acid sequence shown in SEQ ID No. 1 with threonine;

[0021] (6) Replace the phenylalanine at position 210 with leucine, the lysine at position 212 with alanine, the tryptophan at position 217 with glutamine, the isoleucine at position 178 with valine, the histidine at position 224 with arginine, and the leucine at position 236 with isoleucine in the amino acid sequence shown in SEQ ID No.1.

[0022] (7) Replace arginine at position 220 with valine, alanine at position 47 with proline, valine at position 50 with alanine, phenylalanine at position 210 with leucine, lysine at position 212 with alanine, tryptophan at position 217 with glutamine, isoleucine at position 178 with valine, histidine at position 224 with arginine, and leucine at position 236 with isoleucine;

[0023] (8) Replace glutamine at position 239 of the amino acid sequence shown in SEQ ID No.1 with glycine, proline at position 125 with threonine, and serine at position 40 with glutamic acid;

[0024] (9) Replace the following amino acid sequences as shown in SEQ ID No. 1: glutamine at position 239 with glycine, proline at position 125 with threonine, serine at position 40 with glutamic acid, valine at position 124 with threonine, isoleucine at position 127 with glutamic acid, leucine at position 128 with tyrosine, serine at position 72 with asparagine, tyrosine at position 73 with glutamine, threonine at position 76 with valine, lysine at position 195 with alanine, glycine at position 180 with serine, arginine at position 220 with valine, alanine at position 47 with proline, valine at position 50 with alanine, phenylalanine at position 210 with leucine, and lysine at position 212 with alanine.

[0025] (10) the glutamine at position 239, the proline at position 125, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212, the tryptophan at position 217, the isoleucine at position 178, the histidine at position 224, and the leucine at position 236 of the amino acid sequence represented by SEQ ID No. 1 are replaced by glycine, threonine, alanine, serine, valine, proline, alanine, leucine, alanine, glutamine, valine, and isoleucine, respectively;

[0026] (11) the glutamine at position 239 of the amino acid sequence represented by SEQ ID No. 1 is replaced by glycine;

[0027] (12) the glutamine at position 239 and the proline at position 125 of the amino acid sequence represented by SEQ ID No. 1 are replaced by glycine and threonine, respectively;

[0028] (13) the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, and the leucine at position 128 of the amino acid sequence represented by SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, and tyrosine, respectively;

[0029] (14) the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, the threonine at position 76, and the lysine at position 195 of the amino acid sequence represented by SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, serine, and serine, respectively;

[0030] (15) the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, the threonine at position 76, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212, the tryptophan at position 217, the isoleucine at position 178, the histidine at position 224, and the leucine at position 236 of the amino acid sequence represented by SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, serine, serine, alanine, valine, leucine, alanine, glutamine, valine, proline, alanine, leucine, alanine, glutamine, valine, isoleucine, valine, and isoleucine, respectively;

[0031] (16) Replace the following amino acid sequences as shown in SEQ ID No. 1: glutamine at position 239 with glycine, proline at position 125 with threonine, serine at position 40 with glutamic acid, valine at position 124 with threonine, isoleucine at position 127 with glutamic acid, leucine at position 128 with tyrosine, serine at position 72 with asparagine, tyrosine at position 73 with glutamine, threonine at position 76 with valine, and lysine at position 195 with... The following substitutions were made: alanine at position 180 (glycine), serine at position 220 (arginine), valine at position 47 (alanine), proline at position 50 (valine), leucine at position 210 (phenylalanine), leucine at position 212 (lysine), glutamine at position 217 (tryptophan), valine at position 178 (isoleucine), arginine at position 224 (histidine), and isoleucine at position 236 (leucine).

[0032] The above-mentioned imine reductase mutants can be obtained through the following technical methods:

[0033] Using the imine reductase PocIRED with the amino acid sequence shown in SEQ ID No. 1 as the parent, multiple imine reductase mutants with significantly enhanced reductive amination activity were identified through random mutation, combined with high-throughput primary screening and further shake-flask culture screening.

[0034] A third object of the present invention is to provide an isolated nucleic acid that encodes the imine reductase or any imine reductase mutant.

[0035] Furthermore, the nucleic acid sequence encoding the imine reductase PocIRED is shown in SEQ ID No. 3.

[0036] The nucleic acid sequence encoding any imine reductase mutant is a nucleic acid sequence derived from the nucleic acid sequence shown in SEQ ID No. 3 by base substitution, deletion, or insertion of 3 or multiples of 3 bases.

[0037] A fourth objective of this invention is to provide a recombinant expression vector comprising the nucleic acid described in the third objective of this invention.

[0038] In one embodiment of the present invention, the recombinant expression vector is a pET28a plasmid vector containing an editing gene of imine reductase or a mutant thereof.

[0039] A fifth objective of this invention is to provide a recombinant expression transformant comprising the recombinant expression vector.

[0040] In one embodiment of the present application, the recombinant expression transformant is E. coli containing the recombinant expression vector and expressing imine reductase or imine reductase mutant.

[0041] In one embodiment of the present application, the E. coli is E. coli (DE3).

[0042] It is a sixth object of the present application to provide a method for constructing the recombinant expression transformant, comprising the following steps:

[0043] (1) Constructing a recombinant expression vector: inserting imine reductase (IRED) or imine reductase mutant (IRED mutant) gene into Nde I / Hind III enzyme cutting site on pET28a plasmid to obtain recombinant expression vector pET28a-IRED or pET28a-IRED mutant;

[0044] (2) Transforming the recombinant expression vector pET28a-IRED or pET28a-IRED mutant into E. coli expression host to screen positive transformants to obtain the recombinant expression transformant.

[0045] It is a seventh object of the present application to provide a recombinant imine reductase catalyst, which is in any of the following forms:

[0046] (1) Culturing the recombinant expression transformant of the present application to isolate transformant cells containing the imine reductase or imine reductase mutant;

[0047] (2) Culturing the recombinant expression transformant of the present application to isolate crude enzyme solution containing the imine reductase or imine reductase mutant;

[0048] (3) Culturing the recombinant expression transformant of the present application to isolate transformant cells containing the imine reductase or imine reductase mutant, crushing the transformant cells to obtain cell crushing solution;

[0049] (4) Culturing the recombinant expression transformant of the present application to isolate transformant cells containing the imine reductase or imine reductase mutant, crushing the transformant cells to obtain cell crushing solution, and freeze-drying the cell crushing solution to obtain freeze-dried enzyme powder;

[0050] The culture method and conditions of the recombinant expression transformant are conventional methods and conditions in the art for obtaining the recombinant imine reductase catalyst. For example, in one embodiment, the culture method of the recombinant expression transformant comprises the following steps: culturing the recombinant expression transformant of the present application to obtain the recombinant imine reductase mutant. For the recombinant E. coli, the preferred culture medium is LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 6.5-7.0. The preferred culture method is as follows: the recombinant E. coli constructed as described above is inoculated into LB medium containing kanamycin and cultured at 37°C with 180 rpm shaking overnight. The inoculation amount is 1-2% (v / v) into a 500 ml flask containing 100 ml of LB medium (containing kanamycin) and cultured at 37°C with 180 rpm shaking. When the OD600 of the culture solution reaches 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) is added as an inducer at a final concentration of 0.1-0.5 mmol / L, and the culture is induced at 16-25°C for 16-24 h. The culture solution is centrifuged, the precipitate is collected, and then washed twice with physiological saline to obtain the recombinant expression transformant cells. The harvested recombinant cells are freeze-dried to obtain the freeze-dried cells containing the IRED or IRED mutant. The harvested recombinant cells are suspended in 5-10 times the volume (v / w) of buffer, ultrasonically broken, and the supernatant is collected by centrifugation to obtain the crude enzyme solution of the IRED or IRED mutant. The collected crude enzyme solution is frozen at -80°C, and then dried by low-temperature drying using a vacuum freeze dryer to obtain the crude enzyme powder. The obtained crude enzyme powder is stored in a 4°C refrigerator for convenient use. 600

[0051] The eighth object of the present application is to provide the use of the imine reductase or imine reductase mutant or recombinant imine reductase catalyst, in particular, the use of the imine reductase or imine reductase mutant or recombinant imine reductase catalyst in the catalysis of asymmetric reductive amination reaction of racemic ketone substrates to synthesize the chiral amine building block of tofacitinib.

[0052] In one embodiment of the present application, in the catalysis of asymmetric reductive amination reaction of racemic ketone substrates by the imine reductase or imine reductase mutant or recombinant imine reductase catalyst, the coenzyme NADPH is oxidized to NADP + , and the dehydrogenase is used to catalyze the reduction and regeneration of NADP + to NADPH.

[0053] The dehydrogenase is an enzyme catalyst for coenzyme circulation in the catalysis of asymmetric reductive amination reaction of racemic ketone substrates by the imine reductase or imine reductase mutant or recombinant imine reductase catalyst.

[0054] ​In an embodiment of the present application, the dehydrogenase is any one of the following dehydrogenases:

[0055] (1) formate dehydrogenase FDH (Enzyme Microb Tech, 2010, 45: 557-561);

[0056] (2) glucose dehydrogenase GDH (ChemBioChem, 2020, 21: 2680-2688);

[0057] (3) alcohol dehydrogenase ADH (J Am Chem Soc, 1986, 108: 162-169).

[0058] In an embodiment of the present application, the racemic substrate ketone for synthesizing the tofacitinib chiral amine building block is any one of the following compounds:

[0059] In an embodiment of the present application, the substrate is 1-benzyl-4-methylpiperidin-3-one, and the tofacitinib chiral amine building block is 3R, 4R-1-benzyl-N, 4-dimethylpiperidin-3-amine.

[0060] Compared with the prior art, the technical effects of the present application mainly lie in the following aspects:

[0061] The present application provides an imine reductase, an imine reductase mutant, a nucleic acid encoding the imine reductase or the imine reductase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a recombinant imine reductase catalyst, and the application of the recombinant imine reductase catalyst in the preparation of a tofacitinib chiral amine building block.

[0062] The imine reductase and the imine reductase mutant have high expression and can efficiently catalyze the generation of a single configuration chiral amine product from the racemic precursor ketone of tofacitinib through dynamic kinetic resolution and asymmetric amination reaction.

[0063] The imine reductase and the imine reductase mutant catalyze the racemic precursor ketone substrate of the tofacitinib chiral amine building block to perform dynamic kinetic resolution and asymmetric amination reaction by using a method and conditions conventional in the art. In some alternative embodiments, the reaction system includes a substrate racemic ketone (which can be, for example, the substrate 1-benzyl-4-methylpiperidin-3-one) at a concentration of 1-200 g / L, an amine donor (methylamine) at a molar ratio of 1.0-10.0 to the substrate, glucose at a molar ratio of 1.0-2.0 to the substrate, NADP +The addition amount is 0.05-2.0 mmol / L, the molar ratio of the auxiliary substrate glucose to the substrate is 1.0-3.0, the activity ratio of the coenzyme dehydrogenase to the imine reductase is 1.0-3.0, the pH is 5.0-10.0, and the temperature is 20-40 DEG C. During the reaction, intermittent sampling is performed. The conversion rate of the reaction is detected, and the reaction is stopped when the conversion rate reaches more than 90% or the conversion rate no longer continues to increase. The ee value of the product can reach more than 99.9%, and the dr value is greater than 98:2.

[0064] Compared with other chemical or biological catalysts for preparing optically pure tofacitinib chiral amine blocks, the imine reductase and the imine reductase mutant provided by the application have high expression, strong catalytic activity, high stability, good stereoselectivity and wide substrate spectrum, and show wide application prospects in industrial applications. BRIEF DESCRIPTION OF DRAWINGS

[0065] FIG. 1 is a performance characterization result of imine reductase PocIRED mother (WT) and its mutant (M6) catalyzing racemic 1-benzyl-4-methylpiperidin-3-one substrate and its analogs. DETAILED DESCRIPTION

[0066] The application will be described in detail below in combination with the drawings and specific embodiments.

[0067] The reaction or detection conditions described in the content of the application can be combined or changed according to common sense in the art and can be verified by experiments. The technical solutions and technical effects in the application will be described clearly and completely in combination with specific embodiments below, but the protection scope of the application is not limited to these embodiments, and any changes or equivalent substitutions without departing from the concept of the application are included in the protection scope of the application.

[0068] The materials in the following examples are from:

[0069] The plasmid vector pET28a is purchased from Novagen Company.

[0070] The E. coli DH5a and E. coli BL21(DE3) competent cells, 2xTaq PCR MasterMix, and agarose gel DNA recovery kit are purchased from Beijing Tiangen Biotech Co., Ltd.

[0071] The chiral GC analysis method is as follows:

[0072] Instrument: Shimadzu GC-2014; column type: Agilent CP-Chirasil (25 m x 0.25 mm x 0.25 mm); analysis method: 130 DEG C, hold for 30 min, 10 DEG C / min -1to 180℃, hold for 10min;

[0073] High-throughput UPLC analysis method is as follows:

[0074] Instrument: Shimadzu LC-30AD UPLC; Column: Shim-pack GIST (2.1x50mm, 2um); Analysis method: isocratic: A phase (MeOH): B phase (H2O+0.1% diethylamine and 0.05% trifluoroacetic acid) = 8:2; Flow rate: 1.5mL / mL; Column temperature: 40℃; Injection volume: 2μL; Detector: UV detector; Detection wavelength: 205nm.

[0075] Example 1: Screening of imine reductase

[0076] The present embodiment provides an imine reductase PocIRED derived from Pochonia chlamydosporia, which is screened from an imine reductase enzyme library previously constructed in the laboratory of the inventors.

[0077] The amino acid sequence of the imine reductase PocIRED is shown in SEQ ID No. 1. Specifically as follows:

[0078] Example 2: Construction of recombinant expression vector

[0079] The nucleotide sequence of the nucleic acid encoding the PocIRED is shown in SEQ ID No. 3. Specifically as follows:

[0080] The gene encoding PocIRED is inserted into the restriction enzyme cleavage site Nde I / Hind III on the pET28a plasmid by one-step cloning to obtain the recombinant expression vector pET28a-PocIRED.

[0081] The PCR conditions are as follows: 95℃ for 5min; 98℃ for 10s, 55℃ for 5s, 72℃ for 35s (25 cycles); 72℃ for 10min, and then the PCR amplification product is verified and recovered by 1% agarose gel electrophoresis. The specific method of one-step cloning is as follows: the target fragment (0.03pmol) and the Nde I / Hind III linearized vector (0.01pmol) are mixed in the same PCR tube, 5μL of enzyme ClonExpress Mix (2x) purchased from Novozyme is added, ddH2O is added to 10μL, and the reaction is carried out at 50℃ for 15min. The specific primer sequence is as follows:

[0082] Primer 1 (SEQ ID No. 4):

[0083] Primer 2 (SEQ ID No. 5):

[0084] Example 3: Construction of recombinant expression transformant

[0085] The recombinant expression vector pET28a-PocIRED was transformed into E. coli BL21(DE3) using the heat shock method familiar to those skilled in the art, i.e. obtaining the recombinant E. coli BL21(DE3) / pET28a-PocIRED recombinant expression transformant.

[0086] The basic operation of the heat shock method is as follows:

[0087] (1) Take a tube of prepared E. coli BL21(DE3) competent cells and place it in an ice bath.

[0088] (2) Add 1 / 5 volume of recombinant expression vector solution to the competent cell suspension, mix gently, and ice bath for 30 min.

[0089] (3) Place the mixed solution after ice bath in a 42°C pulse heat shock for 90 s, quickly transfer to ice bath for 2 min, add 800 μL of LB medium, and incubate at 37°C for 40 min.

[0090] (4) Take an appropriate amount of transformed competent cell suspension and spread it on an LB plate containing 100 μg / mL kanamycin, and incubate at 37°C for 12-16 h to grow the recombinant expression transformant.

[0091] (5) Pick a single colony for the next experiment.

[0092] Verification of positive clones:

[0093] (1) Randomly pick 10 single colonies from the transformed plate under sterile conditions and place them in Eppendorf tubes containing LB liquid medium (containing kanamycin), and incubate at 37°C, 180 rpm shaking until the medium is turbid.

[0094] (2) Under sterile conditions, take 2 μL of bacterial solution as a template, and use the EasyTaqMix system and pET28a universal T7 primer for amplification.

[0095] (3) Agarose nucleic acid gel electrophoresis detection shows a band at 900 bp, which proves that the clone is a positive clone, and the construction of the recombinant expression transformant E. coli BL21(DE3) / pET28a-PocIRED is successful.

[0096] Blank control: 2 μL of bacterial solution is replaced by sterile water as a template for amplification. Agarose nucleic acid gel electrophoresis detection shows that only a band appears at about 250 bp.

[0097] Example 4: Protein expression analysis of the recombinant expression transformant

[0098] The culture method and culture conditions of the recombinant expression transformant of the present application are not particularly limited, and can be appropriately selected according to the type of host cells and culture methods and other factors according to the ordinary knowledge in the art, as long as the recombinant expression transformant can grow and produce the imine reductase of the present application.

[0099] The culture and protein expression of the recombinant expression transformant are carried out using the E. coli culture system familiar to those skilled in the art.

[0100] The recombinant expression transformant constructed as described above is inoculated into LB medium containing kanamycin and cultured at 37°C with 180 rpm shaking overnight. The inoculation amount is 1% (v / v) into a 500 mL triangular flask containing 100 mL of LB medium (containing kanamycin), and the flask is placed in a 37°C, 180 rpm shaking incubator. When the OD 600 of the culture solution reaches 0.6, isopropyl-β-D-thiogalactoside (IPTG) is added as an inducer at a final concentration of 0.2 mM, and the culture solution is centrifuged after 24 h of induction at 16°C. The precipitate is collected and then washed twice with physiological saline to obtain recombinant expression transformant cells.

[0101] The above 1 g of recombinant expression transformant cells is resuspended in 10 mL of KPB buffer (100 mM, pH 8.0), and an ultrasonic disrupter is used at a power of 400 w for 4 s, with an interval of 6 s, for 15 min in an ice water bath. The broken solution is then centrifuged at 4°C at 12000 rpm and the supernatant is collected. The precipitate is resuspended in 10 mL of KPB buffer (100 mM, pH 8.0). 80 μL of the broken supernatant and the resuspended precipitate are taken in a 1.5 mL Eppendorf tube, mixed with 20 μL of 5×SDS PAGE Loading Buffer, and heated in a boiling water bath or 95°C metal bath for 5 min. The sample is immediately subjected to SDS-PAGE analysis.

[0102] SDS-PAGE results show that the content of the target protein expressed by the recombinant expression transformant constructed in the application can account for more than 50% of the total protein, and 95% of the target protein is soluble expression.

[0103] Example 5: reactivity assay of imine reductase

[0104] An analytical scale enzymatic reaction was performed to determine the reactivity of the imine reductase and its mutants. The determination method and conditions are as follows: a 400 μL reaction volume contains 200 μL of cell-free extracted enzyme solution, 15 mM (final concentration, same below) of racemic 1-benzyl-4-methylpiperidin-3-one substrate, 200 mM methylamine, 1 mM NADP + , 1 mg / mL BmGDH, 22.5 mM glucose, 5% v / v dimethyl sulfoxide, in KPB buffer (100 mM, pH 7.0) at 30°C with 800 rpm stirring for 24 h. Then quench the reaction with 40 μL of 10 M NaOH solution, add 500 μL of ethyl acetate to extract the target reaction, centrifuge to take the supernatant, dry with anhydrous Na2SO4, filter with a filter membrane with a pore size of 0.22 μm, and then perform chiral GC analysis to determine the conversion rate of the substrate and the ee and dr values of the product (conversion: 98%; ee: >99.9%; dr: 99:1).

[0105] Example 6: site-directed saturation mutation of imine reductase PocIRED

[0106] The site-directed saturation mutation of the imine reductase PocIRED gene sequence was performed using the whole plasmid PCR amplification technology, and the template was the recombinant plasmid pET28a-PocIRED of the imine reductase PocIRED.

[0107] The system of the PCR reaction is as follows: 1.0 ng of template, 0.5 μL (10 μM) of each pair of mutant primers, 12.5 μL of PrimeSTAR HS (premix), and adding sterile distilled water to 25 μL.

[0108] The primers of mutant M1 in the following examples are as follows (the primers of other mutants can be introduced by the site-directed mutation method):

[0109] Mutant M1 Primer 1 (SEQ ID No. 6):

[0110] Mutant M1 Primer 2 (SEQ ID No. 7):

[0111] The PCR amplification procedure was as follows: (1) denaturation at 98°C for 10 s; (2) denaturation at 98°C for 10 s, (3) annealing at 55°C for 10 s, (4) extension at 72°C for 7 min, steps (2)-(4) were repeated for 20 cycles, and finally extension at 72°C for 7 min, and preservation at 12°C for 1 h.

[0112] After purification of the PCR amplification product, the PCR amplification product was digested with restriction endonuclease Dpn I at 37°C for 2 h, and then transformed into E. coli BL21(DE3) competent cells, and uniformly spread on LB agar medium plates containing 50 μg / mL kanamycin, and cultured at 37°C overnight to construct a random mutant library. Single clones were selected and cultured in a deep well plate, and each well of the primary plate contained 300 μL of LB medium (containing 50 μg / mL kanamycin) and was cultured at 37°C and 800 rpm overnight. 50 μL of the primary seed liquid was transferred to each well of a secondary plate containing 400 μL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 800 rpm for 3-4 h. 50 μL of 2 mM IPTG (final concentration 0.2 mM) was added to each well for induction, and the culture was continued at 20°C for 16-18 h. The deep well plate was centrifuged at 3500 rpm and 4°C for 10 min, and the supernatant was discarded. 200 μL of lysis solution (Tris-Hcl solution (100 mM, pH 9.0) containing 1.5 g / L lysozyme and 0.01 mg / L DNase I enzyme) was added to each well, and the cells were fully suspended by shaking, and incubated at 37°C for 1 h. 200 μL of Tris-Hcl solution (100 mM, pH 9.0) was added to each well, and centrifuged at 3500 rpm and 4°C for 15 min.

[0113] The mutant screening method was as follows:

[0114] 190 μL of the above-mentioned lysis solution was taken from each well after centrifugation, and the supernatant was transferred to a new deep well plate. 1 mM NADP + , 75 mM glucose, 20% (v / v) DMSO, 3 mg / mL crude enzyme powder of glucose dehydrogenase, 50 mM racemic 1-benzyl-4-methylpiperidin-3-one, and 200 mM methylamine hydrochloride, and the volume of each well was made up to 400 μL with Tris-Hcl solution (100 mM, pH 9.0), and the reaction was carried out at 37°C and 800 rpm for 24 h. The reaction was terminated by adding 1200 μL of methanol to each well, and shaken at 800 rpm for 5 min. Then the deep well plate was centrifuged at 3,500 rpm and 4°C for 15 min. 200 μL was taken from each well and transferred to an enzyme-labeled plate, and the conversion rate was analyzed by UPLC.

[0115] Example 7: Activity determination of imine reductase PocIRED and its mutants

[0116] The assay system was 400 μL of Tris-Hcl solution (100 mM, pH 9.0) containing 50 mM racemic 1-benzyl-4-methylpiperidin-3-one, 1000 mM methylamine, 1 mM NADP + , 200 mM glucose, 15% (v / v) DMSO, imine reductase or mutant lysate supernatant after centrifugation, and 2 mg / mL of crude enzyme powder of glucose dehydrogenase, and the conversion rate at the initial stage of the reaction was measured under the conditions of 33°C and 800 rpm. The reaction was terminated by adding 30 μL of 10 M sodium hydroxide solution per well, and extraction was performed using 500 μL of methyl tert-butyl ether. The organic phase was dried using anhydrous magnesium sulfate, and the conversion rate was measured using gas chromatography. The specific enzyme activity was calculated based on the conversion rate. The results are shown in Table 1.

[0117] Table 1 Enzyme activity of PocIRED and its mutants

[0118] In Table 1, WT refers to PocIRED,

[0119] M1 refers to the mutant (11) in the summary, i.e., the glutamine at position 239 of the amino acid sequence shown in SEQ ID No. 1 is replaced with glycine;

[0120] M2 refers to the mutant (12) in the summary, i.e., the glutamine at position 239 of the amino acid sequence shown in SEQ ID No. 1 is replaced with glycine, and the proline at position 125 is replaced with threonine;

[0121] M3 refers to the mutant (13) in the summary, i.e., the glutamine at position 239 of the amino acid sequence shown in SEQ ID No. 1 is replaced with glycine, the proline at position 125 is replaced with threonine, the serine at position 40 is replaced with glutamic acid, the valine at position 124 is replaced with threonine, the isoleucine at position 127 is replaced with glutamic acid, and the leucine at position 128 is replaced with tyrosine;

[0122] M4 refers to the mutant (14) in the summary, i.e., the glutamine at position 239 of the amino acid sequence shown in SEQ ID No. 1 is replaced with glycine, the proline at position 125 is replaced with threonine, the serine at position 40 is replaced with glutamic acid, the valine at position 124 is replaced with threonine, the isoleucine at position 127 is replaced with glutamic acid, the leucine at position 128 is replaced with tyrosine, the serine at position 72 is replaced with asparagine, the tyrosine at position 73 is replaced with glutamine, the threonine at position 76 is replaced with valine, and the lysine at position 195 is replaced with alanine;

[0123] M5 refers to mutant (15) in the summary of the invention, i.e. the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, the threonine at position 76, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50 of the amino acid sequence shown in SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, serine, valine, proline, alanine, respectively;

[0124] M6 refers to mutant (16) in the summary of the invention, i.e. the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, the threonine at position 76, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212, the tryptophan at position 217, the isoleucine at position 178, the histidine at position 224, the leucine at position 236 of the amino acid sequence shown in SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, serine, valine, proline, alanine, respectively. The amino acid sequence of M6 is shown in SEQ ID NO. 2, as follows:

[0125] Negative control refers to blank reaction without any imine reductase or mutant of the present application.

[0126] Example 8: Preparation of crude enzyme solution of imine reductase PocIRED and its mutants

[0127] The recombinant E. coli constructed as described above was inoculated into TB medium containing kanamycin and cultured at 37℃ with 180 rpm shaking overnight. The inoculation amount was 1% (v / v) into a 500 mL flask containing 100 mL of TB medium (containing kanamycin) and cultured at 37℃ with 180 rpm shaking. When the OD600 of the culture solution reached 0.6-0.8, 0.5 mM IPTG was added to induce the expression of the recombinant protein. After induction for 4-6 hours, the culture solution was centrifuged at 8000 rpm for 10 minutes to obtain the cell pellet. The cell pellet was resuspended in 50 mM Tris-HCl buffer (pH 8.0) and sonicated for 10 minutes to obtain the crude enzyme solution. The crude enzyme solution was centrifuged at 8000 rpm for 10 minutes to obtain the supernatant, which was used as the enzyme solution for the following experiments. 600When the OD600 reached 0.6, IPTG was added to a final concentration of 0.2 mM as an inducer, and the culture was induced at 16°C for 24 h. The culture was centrifuged to collect the precipitate, which was then washed twice with physiological saline to obtain the recombinant expression transformant cells. The harvested recombinant cells were suspended in 10 times the volume (v / w) of buffer, and were subjected to ultrasonic disruption. The supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant imine reductase PocIRED or its mutant.

[0128] Example 9: Preparation of crude enzyme powder of imine reductase PocIRED and its mutants

[0129] The crude enzyme solution of imine reductase obtained in Example 8 was frozen at -80°C, and then was subjected to low-temperature drying using a vacuum freeze dryer to obtain crude enzyme powder of imine reductase or its mutants. The obtained crude enzyme powder was stored in a 4°C refrigerator, and could be conveniently used.

[0130] Example 10: PocIRED mutant M6 catalyzes synthesis of a chiral amine building block of tofacitinib

[0131] A preparation reaction with a volume of 500 mL was performed in a 1 L reactor to test the production performance of the catalytic system. The specific operation was as follows. 200 mL of water and 7.5 g of glycine were added to the reactor, and after being stirred uniformly, the pH was adjusted to 9.5 using a 10 M sodium hydroxide solution. Subsequently, 33.5 g of methylamine hydrochloride (1000 mM, 1.8 eq.) and 65 g of glucose (1.3 eq.) were added to the reaction system and stirred uniformly. 56 g of 1-benzyl-4-methylpiperidin-3-one substrate (550 mM, 110 g / L) was dissolved in 75 mL of cosolvent dimethyl sulfoxide (DMSO, 15% v / v) and added to the reactor. 0.4 g of coenzyme NADP +(1 mM), 0.4 g of BmGDH lyophilized enzyme powder (0.8 mg / mL), and 5 g of PocIRED-M6 mutant lyophilized enzyme powder (10.0 mg / mL). Water was added to make up the 500 mL reaction volume. The reaction mixture was stirred at 30 °C and 600 rpm for 24 h, and the pH of the reaction system was maintained at 9.5 by titration with 1.5 M aqueous methylamine during the reaction. During this time, samples were taken for GC-MS detection of the reaction conversion rate, and the reaction conversion rate reached 93% after 24 h. Acetic acid was added to quench the reaction system to pH 3.0 and stirred for 10 min. 25 g of diatomite was added, and the mixture was stirred for 30 min to adsorb the denatured protein precipitated by the acid. The reaction mixture was filtered to remove the diatomite-protein complex and other insoluble impurities, and the filter cake was washed with 1 M hydrochloric acid and combined with the reaction mixture. The impurities in the reaction mixture were removed by extraction with dichloromethane (1 L x 4). The pH of the reaction mixture was adjusted to 11-12 with 10 M sodium hydroxide solution, and the target product was extracted with ethyl acetate (1 L x 3). The extracted organic phase was collected and combined, dried over anhydrous Na2SO4, and then rotary evaporated under reduced pressure to obtain 49.5 g of brown oil (74% yield, >99.9% ee, and 98:2 dr).

[0132] Example 11: Reaction performance determination of imine reductase PocIRED parent (WT) and its mutant (M6) on different substrates

[0133] The imine reductase PocIRED parent (WT) and its mutant (M6) catalyze the racemic 1-benzyl-4-methylpiperidin-3-one substrate and its analogs to harvest a series of structurally similar chiral amine building blocks, and the chiral amine building blocks can be used to synthesize the drug tofacitinib. The specific reaction performance characterization is shown in FIG. 1.

[0134] The above description of the embodiments is to enable any person skilled in the art who can understand and use the present application. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art within the scope of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A mutant of an imine reductase, characterized in that, It is a derivative protein of a new amino acid sequence formed by replacing one or more of the amino acid residues at positions 239 glycine, 125 proline, 40 serine, 124 valine, 127 isoleucine, 128 leucine, 72 serine, 73 tyrosine, 76 threonine, 195 lysine, 180 glycine, 220 arginine, 47 alanine, 50 valine, 210 phenylalanine, 212 lysine, 217 tryptophan, 178 isoleucine, 224 histidine or 236 leucine of the amino acid sequence shown as SEQ ID No. 1 with other amino acid residues.

2. The imine reductase mutant of claim 1, wherein, The imine reductase mutant has one of the following sequences: (1) replacing the 239th glutamine, the 125th proline, the 40th serine of the amino acid sequence shown as SEQ ID No. 1 with glycine, threonine, glutamic acid, respectively; (2) replacing the 72nd serine, the 73rd tyrosine, the 76th threonine, the 195th lysine, the 180th glycine of the amino acid sequence shown as SEQ ID No. 1 with asparagine, glutamine, valine, alanine, serine, respectively; (3) replacing the 125th proline, the 40th serine, the 124th valine, the 127th isoleucine, the 128th leucine, the 72nd serine, the 73rd tyrosine, the 76th threonine of the amino acid sequence shown as SEQ ID No. 1 with threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, respectively; (4) replacing the 239th glutamine, the 125th proline, the 40th serine, the 124th valine, the 127th isoleucine, the 128th leucine, the 72nd serine, the 73rd tyrosine, the 76th threonine, the 195th lysine, the 180th glycine, the 220th arginine, the 47th alanine, the 50th valine, the 210th phenylalanine, the 212th lysine, the 217th tryptophan, the 178th isoleucine, the 224th histidine or the 236th leucine of the amino acid sequence shown as SEQ ID No. 1 with other amino acid residues to form a new amino acid sequence; (5) replacing the 125th proline of the amino acid sequence shown as SEQ ID No. 1 with threonine; (6) replacing the 210th phenylalanine, the 212th lysine, the 217th tryptophan, the 178th isoleucine, the 224th histidine or the 236th leucine of the amino acid sequence shown as SEQ ID No. 1 with leucine, alanine, glutamine, valine, arginine, isoleucine, respectively. (7) the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212, the tryptophan at position 217, the isoleucine at position 178, the histidine at position 224, the leucine at position 236 of the amino acid sequence shown in SEQ ID No. 1 is replaced by valine, proline, alanine, leucine, alanine, glutamine, valine, arginine, isoleucine, respectively; (8) the glutamine at position 239, the proline at position 125, the serine at position 40 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine, threonine, glutamic acid, respectively; (9) the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128, the serine at position 72, the tyrosine at position 73, the threonine at position 76, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, leucine, alanine, respectively; (10) the glutamine at position 239, the proline at position 125, the lysine at position 195, the glycine at position 180, the arginine at position 220, the alanine at position 47, the valine at position 50, the phenylalanine at position 210, the lysine at position 212, the tryptophan at position 217, the isoleucine at position 178, the histidine at position 224, the leucine at position 236 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine, threonine, alanine, serine, valine, proline, alanine, leucine, alanine, respectively; (11) the glutamine at position 239 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine; (12) the glutamine at position 239, the proline at position 125 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine, threonine, respectively; (13) the glutamine at position 239, the proline at position 125, the serine at position 40, the valine at position 124, the isoleucine at position 127, the leucine at position 128 of the amino acid sequence shown in SEQ ID No. 1 is replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, respectively. (14) the 239th glutamine, the 125th proline, the 40th serine, the 124th valine, the 127th isoleucine, the 128th leucine, the 72nd serine, the 73rd tyrosine, the 76th threonine, the 195th lysine of the amino acid sequence shown in SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, respectively; (15) the 239th glutamine, the 125th proline, the 40th serine, the 124th valine, the 127th isoleucine, the 128th leucine, the 72nd serine, the 73rd tyrosine, the 76th threonine, the 195th lysine of the amino acid sequence shown in SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, respectively, the 180th glycine is replaced by serine, the 220th arginine is replaced by valine, the 47th alanine is replaced by proline, the 50th valine is replaced by alanine; (16) the 239th glutamine, the 125th proline, the 40th serine, the 124th valine, the 127th isoleucine, the 128th leucine, the 72nd serine, the 73rd tyrosine, the 76th threonine, the 195th lysine of the amino acid sequence shown in SEQ ID No. 1 are replaced by glycine, threonine, glutamic acid, threonine, glutamic acid, tyrosine, asparagine, glutamine, valine, alanine, respectively, the 180th glycine is replaced by serine, the 220th arginine is replaced by valine, the 47th alanine is replaced by proline, the 50th valine is replaced by alanine, the 210th phenylalanine is replaced by leucine, the 212th lysine is replaced by alanine, the 217th tryptophan is replaced by glutamine, the 178th isoleucine is replaced by valine, the 224th histidine is replaced by arginine, the 236th leucine is replaced by isoleucine.

3. An isolated nucleic acid, comprising, The nucleic acid encodes the imine reductase mutant as claimed in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as claimed in claim 3.

5. A recombinant expression transformant, characterized by, The recombinant expression transformant comprises the recombinant expression vector as claimed in claim 4.

6. A recombinant imine reductase mutant catalyst characterized in that, The recombinant imine reductase mutant catalyst is in any of the following forms: (1) culturing the recombinant expression transformant as claimed in claim 5, and isolating the transformant cells containing the imine reductase mutant; (2) culturing the recombinant expression transformant as claimed in claim 5, and isolating the crude enzyme solution containing the imine reductase mutant; (3) drying the crude enzyme solution containing the imine reductase mutant to obtain the crude enzyme powder.

7. A process for the enzymatic conversion to prepare a chiral amine building block for tofacitinib, characterized in that, The chiral amine building block of tofacitinib is prepared by dynamic kinetic resolution-asymmetric reductive amination of racemic precursor ketone using the imine reductase mutant of claim 1 or 2 or the recombinant imine reductase mutant catalyst of claim 6 as catalyst in the presence of coenzyme NADPH.

8. The method of claim 7, wherein, coupling the dehydrogenation reaction catalyzed by dehydrogenase, NADP + is reduced and regenerated to NADPH by enzymatic method; during the reaction, the coenzyme NADPH is oxidized to NADP + +; NADP + + is reduced and regenerated to NADPH by dehydrogenase catalysis; The dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase.

9. The method of claim 8, wherein, The reaction conditions are: the concentration of the substrate racemic precursor ketone is 1-200 g / L, the molar ratio of glucose to substrate is 1.0-2.0, the concentration of NADP + The addition amount is 0.05-2.0 mmol / L, the pH is 4.0-10.0, and the temperature is 20-40℃.

10. The method of claim 7, wherein, The substrate for the synthesis of the chiral amine building block of tofacitinib is selected from the group consisting of compounds represented by any one of the following chemical structures:

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