Imine reductase mutants and their applications in the synthesis of chiral intermediates of larotrectinib

Abstract

The present invention relates to imine reductase mutants and their applications in the synthesis of larotrectinib chiral intermediates, and belong to the field of bioengineering technology. The specific activity of the imine reductase mutant of the present invention is as high as 19.2U / mg, and it is possible to efficiently catalyze the asymmetric reduction of 2-(2,5-difluorophenyl) pyrroline, synthesize the chiral intermediate (R)-2-(2,5-difluorophenyl) pyrrolidines of larotrectinib, and the substrate concentration in the enzymatic reaction is as high as 80g / L, and the product space-time yield is as high as 350g / L / d, and the optical purity is more than 99.5%. In addition, the imine reductase mutant also has high catalytic activity for some 2-aryl-substituted pyrrolines, and the optical purity of the product is all up to more than 99%. The mutant of the present invention has extremely high catalytic activity, substrate tolerance and space-time yield, and has strong industrial application prospects in the synthesis of chiral 2-aryl pyrrolidines, especially larotrectinib chiral intermediates (R)-2-(2,5-difluorophenyl) pyrrolidines.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to an imine reductase mutant and a gene encoding the same, a recombinant expression vector and a recombinant expression transformant comprising the imine reductase mutant gene, a method for preparing the recombinant imine reductase mutant, and its application in catalyzing the asymmetric reduction of 2-(2,5-difluorophenyl)pyrroline and its analogs to prepare a chiral intermediate of larotrectinib ( R )-2-(2,5-difluorophenyl)pyrrolidine and chiral 2-arylpyrrolidine. Background Art

[0002] Chiral 2-arylpyrrolidine is a nitrogen heterocyclic chiral amine building block that is widely used in the synthesis of drug molecules. For example, the compound LY2456302 developed by Eli Lilly is a high affinity and high selectivity κ Opioid receptor antagonists, which block the body's own κ Stimulation mediated by agonists or exogenous agonists such as morphine, as opposed to μ and δ Opioid receptors, with more than 30-fold functional selectivity, can be used in addiction treatment and anti-major depression treatment. Another compound, LY27950503, is also a (KOR) PET radiosensor. κ Opioid receptor antagonists have similar effects. The myosin-related kinase (TRK) receptor family is involved in neuronal development. When abnormal fusion occurs, oncogenic TRK fusions induce cancer cell proliferation and participate in key downstream cancer-related signaling pathways. Larotrectinib (LOXO-101) is the first solid tumor anticancer drug targeting the ATP binding site of the TRK receptor family. Its molecular structure is shown below. This drug molecule was launched in the United States in 2018.

[0003]

[0004] Larotrectinib structure

[0005] Regarding the synthesis of chiral 2-substituted pyrrolidines, Helmchen utilized a rhodium catalyst (Synlett, 2009, 4: 1413-1416) to carry out asymmetric hydrogenation reaction of allylamine in 2009, thereby synthesizing various chiral 2-substituted pyrrolidines. Zhang utilized Ir-f-Binaphane catalyst (Advanced Synthesis & Catalysis, 2010, 352: 3121-3125) to reduce a series of 2-aryl pyrrolines, generating various chiral 2-aryl pyrrolidines. However, these chemical processes not only use heavy metal catalysts such as iridium, rhodium and ruthenium, which result in a significant increase in removal costs, but also require complex protection and deprotection processes during the reaction. Therefore, it is urgent to develop a more environmentally friendly, efficient and safe synthetic method.

[0006] Enzymatic asymmetric reduction methods have many advantages, such as high selectivity, mild reaction conditions, and simple operation. Therefore, the enzymatic asymmetric reduction of imines to synthesize chiral amines has attracted increasing attention in recent years. For the enzymatic synthesis of 2-substituted pyrrolidines, the Turner group (ChemCatChem, 2015, 7: 579-583; ChemCatChem, 2013, 5: 3505-3508) screened for imine reductases with R and S selectivity, respectively. R IR and S IR, but as the substituent becomes larger, the enzyme selectivity and catalytic activity become lower, and it cannot meet the synthesis needs of the sterically hindered substrate 2-arylpyrrolidine. In 2016, the Turner group (ACS Catalysis, 2016, 6: 3880-3889) identified a natural source S-selective imine reductase Ao IREDs catalyze the reduction of 2-substituted pyrrolines. While they exhibit high catalytic efficiency for the less sterically hindered 2-methylpyrroline, they exhibit lower catalytic activity for pyrrolines substituted with bulky substituents, such as 2-aryl and p-fluorophenyl groups. Recently, screening for 2-aryl-substituted pyrroline substrates has yielded several natural imine reductases (Organic Letters, 2020, 22:3367-3372, Org. Process Res. Dev. 2022, 26, 2067–2074). These enzymes have achieved asymmetric reductions of a range of 2-aryl pyrroline substrates, but they also suffer from low activity and substrate concentration. While imine reductases have made significant progress in catalyzing asymmetric reductions in recent years, most natural enzymes suffer from substrate inhibition and low catalytic activity, resulting in low substrate loading. Reactions with high substrate concentrations require the addition of large amounts of enzyme, making post-processing cumbersome and costly, limiting their industrial application.

[0007] In the previous research, the inventors Streptomyces clavuligerus Imine reductase Sc IR, which can catalyze the asymmetric hydrogenation of 2-(2,5-difluorophenyl)pyrroline and various 2-arylpyrrolines with high stereoselectivity to prepare the corresponding larotrectinib chiral intermediates ( R )-2-(2,5-difluorophenyl)pyrrolidine and chiral 2-arylpyrrolidines ( Org. Lett. , 2017, 19, 3151–3154). However, the enzyme has problems such as low activity, poor thermal stability, and inability to convert high-concentration substrates. For example, its specific activity against 2-(2,5-difluorophenyl)pyrroline is only 0.18 U / mg, and the substrate concentration is only 2 g / L. Summary of the Invention

[0008] The purpose of the present invention is to overcome the problems and shortcomings of the prior art and to modify the wild-type imine reductase by using protein engineering techniques such as site-directed mutagenesis and random mutagenesis. Sc IR was used for molecular modification to obtain an imine reductase mutant with significantly improved catalytic activity, thermal stability and substrate tolerance, thereby increasing the substrate loading capacity in the reaction. R This study not only provides an efficient and green enzymatic synthesis of )-2-(2,5-difluorophenyl)pyrrolidine, but also provides a green and efficient enzymatic synthesis process for chiral 2-arylpyrrolidines, and will also accelerate the application of imine reductase in industry.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] One of the technical solutions adopted by the present invention is to provide an imine reductase with improved catalytic activity, stability and substrate tolerance to 2-(2,5-difluorophenyl)pyrroline Sc IR mutants.

[0011] For the imine reductase having the amino acid sequence shown in SEQ ID No.2 Sc IR is mutated to replace one or more amino acid residues at position 122 valine, position 177 phenylalanine, position 169 methionine, position 185 tryptophan, position 211 serine, position 270 methionine, position 214 glycine, position 45 glutamic acid, position 53 aspartic acid and position 265 phenylalanine with other amino acid residues to form a derivative protein with a new amino acid sequence.

[0012] Among them, editing imine reductase Sc The nucleotide sequence of the IR gene is shown in SEQ ID No.1.

[0013] Wherein, SEQ ID No.1 sequence is as follows:

[0014] aacgagctgg tcgtgctgag cctgaccgac tacgacgcga tgtacgcgct gctcgggccc 240

[0015] gccgccgacg cgctcgccgg aaaggtcgtc gtcaacctca gctccgacac cccggagaag 300

[0016] acccgcgcgg gcgcccgctg gatcgcggag cacggcggca ctctgatcgc gggcggcgtc 360

[0017] accgtaccgc cgtcgggcat cggttcgccc gagtcgtccg cgttctacag cggtccgtcg 420

[0018] gcggcgttcg agcgccaccg ggaaacgctc cggacgctga cccgcaccga ctaccggggc 480

[0019] gaggacccgg gtctcgcggc gctgatgtac cagatcggga tggtgatgtt ctggaacgcg 540

[0020] atgctcggct actggcaggc ggtggccctg gccgacgcca acggcctcaa ggcggcggac 600

[0021] atcctgccgc acgcctcgga caccgtggcc tcgctcccgg ggttcctccg cttctacgcc 660

[0022] gaccgcatcg acacggggca ccacgggggc gatgtggacc ggctggccat gggcaccgcg 720

[0023] agcgtcgagc acatcctgca caccatggcc gactccgggg tcgacaccgc gctccccgag 780

[0024] gcggtcgtgg cgttcttccg gcggggcatg gcggcgggct acgcggagaa cagcttctcc 840

[0025] agcatggtgg agctgctgaa gaagccgtcc tga 873

[0026] SEQ ID No. 2 is as follows:

[0027] MSRPAPLTLI GLGPMGQAMG NALLDRGHGL TVWNRTASRA DALVERGAVR APDVAAAVAA 60

[0028] NELVVLSLTD YDAMYALLGP AADALAGKVV VNLSSDTPEK TRAGARWIAE HGGTLIAGGV 120

[0029] TVPPSGIGSP ESSAFYSGPS AAFERHRETL RTLTRTDYRG EDPGLAALMY QIGMVMFWNA 180

[0030] MLGYWQAVAL ADANGLKAAD ILPHASDTVA SLPGFLRFYA DRIDTGHHGG DVDRLAMGTA 240

[0031] SVEHILHTMA DSGVDTALPE AVVAFFRRGM AAGYAENSFS SMVELLKKPS 290

[0032] Targeting imine reductase Sc IR is subjected to random mutation or single-point saturation mutation, and the activity is detected using the substrate 2-(2,5-difluorophenyl)pyrroline to screen mutants with significantly improved activity and thermal stability. Specifically, the imine reductase mutant is a wild-type imine reductase Sc Mutants obtained by causing any of the following mutations in the amino acid sequence of IR:

[0033] (1) replacing valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 with cysteine;

[0034] (2) replacing the valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 with cysteine, and replacing the phenylalanine at position 177 with glutamic acid, arginine, or tryptophan;

[0035] (3) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 122 is replaced by cysteine, the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; the methionine at position 169 is replaced by phenylalanine or tryptophan, the tryptophan at position 185 is replaced by leucine or isoleucine, the serine at position 211 is replaced by alanine, glycine or valine, and the methionine at position 270 is replaced by glutamic acid, arginine or tryptophan;

[0036] (3) in the amino acid sequence shown in SEQ ID No. 2, the valine at position 122 is replaced by cysteine, the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; the methionine at position 169 is replaced by phenylalanine or tryptophan, the tryptophan at position 185 is replaced by leucine or isoleucine, the serine at position 211 is replaced by alanine, glycine or valine, and the methionine at position 270 is replaced by glutamic acid, arginine or tryptophan;

[0037] (4) The valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 is replaced by cysteine, the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; the methionine at position 169 is replaced by phenylalanine or tryptophan, the tryptophan at position 185 is replaced by leucine or isoleucine, the serine at position 211 is replaced by alanine, glycine or valine, the methionine at position 270 is replaced by glutamic acid, arginine or tryptophan; and the glycine at position 214 is replaced by phenylalanine or tryptophan;

[0038] (5) The valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 is replaced by cysteine, the phenylalanine at position 177 is replaced by glutamic acid, arginine or tryptophan; the methionine at position 169 is replaced by phenylalanine or tryptophan, the tryptophan at position 185 is replaced by leucine or isoleucine, the serine at position 211 is replaced by alanine, glycine or valine, the methionine at position 270 is replaced by glutamic acid, arginine or tryptophan; the glycine at position 214 is replaced by phenylalanine or tryptophan; the glutamic acid at position 45 is replaced by alanine or glycine, the aspartic acid at position 53 is replaced by threonine or tryptophan, and the phenylalanine at position 265 is replaced by alanine, leucine or isoleucine.

[0039] The method for obtaining the mutant is specifically as follows:

[0040] Based on Streptomyces clavuligerus Wild-type imine reductase Sc The gene sequence of IR was used as a template, and a random mutation library was constructed using an error-prone PCR strategy. High-throughput screening was performed on each round of constructed mutation libraries to obtain the above-mentioned various mutants.

[0041] The error-prone PCR amplification for constructing the random mutation library is a conventional technique in the art. The PCR amplification reaction system is as follows: 0.5-20 ng template, 1.5 μl (10 μM) of each pair of mutation primers, 8.75 μl MnCl2 (1 mM), 25 μl 2 × Taq mix, and sterile distilled water to 50 μl.

[0042] The error-prone PCR amplification procedure was as follows: (1) denaturation at 95°C for 3 min; (2) denaturation at 95°C for 30 s, (3) annealing at 55°C for 30 s, and (4) extension at 72°C for 1 min. Steps (2) to (4) were repeated for 30 cycles, followed by a final extension at 72°C for 10 min. The product was then stored at 12°C.

[0043] The second technical solution adopted by the present invention is to provide an isolated nucleic acid encoding the imine reductase mutant.

[0044] The preparation method of the nucleic acid of the present invention is a conventional preparation method in the art, which comprises: obtaining a nucleic acid molecule encoding the imine reductase mutant by gene cloning technology, or obtaining a nucleic acid molecule encoding the imine reductase mutant by artificial full sequence synthesis.

[0045] The preparation method of the nucleic acid of the present invention is a conventional preparation method in the art. The preferred preparation method is: using the mutant DNA sequence obtained in technical solution 1 as a template, PCR amplification of the desired target gene is performed to obtain the nucleic acid molecule with a point mutation.

[0046] The PCR amplification technique is conventional in the art. An optional PCR amplification system is: 0.5-20 ng template, 1 μl of each pair of amplification primers (10 μM), 10 μl 2 × Primestar mix, and sterile distilled water to 20 μl.

[0047] An alternative PCR amplification program is: (1) denaturation at 98°C for 10 s; (2) denaturation at 98°C for 10 s, (3) annealing at 55°C for 15 s, (4) extension at 72°C for 6.5 min, steps (2) to (4) are performed for a total of 25 cycles, and the final extension is at 72°C for 10 min, and the product is stored at 12°C.

[0048] The third technical solution adopted by the present invention is to provide a recombinant expression vector comprising the above-mentioned nucleic acid.

[0049] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the imine reductase mutant gene of the present invention to various commercially available expression vectors. The expression vector of the present invention is preferably a 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 the expression vector pET-28a are respectively treated with restriction endonucleases Bam HI and There The residues were double-digested with d III to form complementary sticky ends, which were then ligated with T4 DNA ligase to form a recombinant expression plasmid containing the imine reductase gene of the present invention.

[0050] The fourth technical solution adopted by the present invention is to provide a recombinant expression transformant comprising the above-mentioned recombinant expression vector.

[0051] The method for preparing the recombinant expression transformant is preferably: transforming the above-mentioned recombinant expression vector into a host microorganism. The host microorganism is preferably: Escherichia coli ( Escherichia coli , abbreviated as E. coli ), preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5α. The aforementioned recombinant expression plasmid is transformed into Escherichia coli E. coli BL21(DE3) was used to obtain the preferred genetically engineered strain of the present invention. Plasmid transformation methods can be conventional in the art, such as electroporation and heat shock. Heat shock is preferably used for transformation. The plasmid solution is mixed with competent cells, heat-shocked at 42°C for 90 seconds, then ice-bathed for 3 minutes, and then revived at 37°C for 1 hour. The cells are then plated on LB agar containing kanamycin and cultured to obtain the target recombinant expression transformants.

[0052] The fifth technical solution adopted by the present invention: provides a method for preparing an imine reductase mutant, comprising the following steps: culturing the recombinant expression transformant, and obtaining the recombinant imine reductase mutant from the culture.

[0053] The method for preparing the recombinant imine reductase mutant is preferably as follows: the recombinant Escherichia coli is inoculated into LB medium (yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.0) containing kanamycin (50 μg / mL), cultured at 37°C with shaking overnight, and the mixture is diluted with 1% ( v / v ) was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium and cultured in a shaking incubator at 37°C and 180 rpm. 600When the p-value reached 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM for induction at 25°C. After 24 hours of induction, the culture medium was centrifuged and washed twice with saline to obtain resting cells. The resulting resting cells were suspended in potassium phosphate buffer (100 mM KPB, pH 7.0), disrupted by sonication in an ice-water bath, and the supernatant was collected by centrifugation to obtain the crude recombinant enzyme solution. Analysis of the crude enzyme solution by polyacrylamide gel electrophoresis showed that the recombinant protein was fully soluble in the cells.

[0054] The catalyst of the present invention that catalyzes the asymmetric reduction reaction of 2-(2,5-difluorophenyl)pyrroline and its analogs to form optically active (R)-2-(2,5-difluorophenyl)pyrrolidine and its analogs can be the above-mentioned recombinant imine reductase mutant protein, or a recombinant expression transformant resting cell containing the recombinant imine reductase mutant.

[0055] The sixth technical solution adopted by the present invention is to provide the use of the imine reductase mutant or the recombinant Escherichia coli whole cell as a catalyst to catalyze the asymmetric reduction reaction of 2-arylpyrroline derivatives to form chiral amines.

[0056] The 2-arylpyrroline derivative is selected from one or more of the structures A shown in the following structural formulas 1a-11:

[0057] .

[0058] In the application of the imine reductase mutant or the recombinant Escherichia coli whole cell of the present invention as a catalyst for catalyzing the asymmetric reduction reaction of 2-arylpyrroline derivatives to form chiral amines, the 2-arylpyrroline derivative is used as a substrate, and in the presence of the coenzyme NADPH, the imine reductase mutant or the recombinant Escherichia coli whole cell is used to catalyze the asymmetric reduction of the 2-arylpyrroline derivative to prepare optically active (R)-2-(2,5-difluorophenyl)pyrrolidine and its analogs, and NADPH is simultaneously oxidized to generate NADP. + .

[0059] During the reaction, the glucose dehydrogenation reaction catalyzed by glucose dehydrogenase is coupled to convert NADP + Enzymatic reduction regenerates it to NADPH.

[0060] The conditions for the asymmetric reduction reaction of the present invention can be selected according to the conventional conditions of such reactions in the art. The preferred application is: the reaction is carried out in an aqueous phase, the concentration of the substrate is 2-80 g / L, and the concentration of the cosolvent DMSO is 0-5% ( w / v), the dosage of the imine reductase mutant resting cells is 0.1~10 kU / L, and the coenzyme NADP is additionally added + The concentration of is 0~0.5 mM, the concentration of cosubstrate glucose is 20~700 mM, the amount of glucose dehydrogenase added is 0.1~10 kU / L, the reaction pH is 6.0~8.0, and the reaction temperature is 20~40°C.

[0061] The multiple imine reductase mutants of the present invention are suitable for catalyzing the reduction of 2-(2,5-difluorophenyl)pyrroline and its analogues to prepare optically pure (R)-2-(2,5-difluorophenyl)pyrrolidine and its analogue chiral drug intermediates.

[0062] Compared with the prior art, the present invention has the following advantages and effects: the imine reductase mutant of the present invention has higher catalytic activity and thermal stability, and the catalytic activity for 2-(2,5-difluorophenyl)pyrroline substrate is as high as 19.2 U / mg, which is much higher than that of the currently reported imine reductases, and can efficiently catalyze the asymmetric reduction of 2-(2,5-difluorophenyl)pyrroline to synthesize the chiral intermediate of larotrectinib ( R )-2-(2,5-difluorophenyl)pyrrolidine. The enzymatic reaction achieved substrate concentrations as high as 80 g / L, a product space-time yield of 350 g / L / d, and an optical purity exceeding 99.5%. These substrate concentrations and space-time yields significantly exceed those of previously reported imine reductase-catalyzed processes. Compared to metal-catalyzed processes, the enzymatic reaction described in this invention offers advantages such as high product optical purity, mild reaction conditions, safe operation, and an environmentally friendly process, demonstrating promising industrial applications. DETAILED DESCRIPTION

[0063] The present invention is described in detail below with reference to specific embodiments.

[0064] Example 1 Imine Reductase Sc Random mutation of IR

[0065] Imine reductase PCR Sc Random base mutations were introduced into the IR gene sequence using the following primers:

[0066] Upstream primer (as shown in SEQ ID No.3):

[0067] 5'-AACTTTAAGAAGGAGATATACCATGGGCCATCATCATCATCACATGTCCCGCCCCGCGCCCCTCACC-3'

[0068] Downstream primer (as shown in SEQ ID No.4):

[0069] 5'-GTGGTGGTGCTCGAGTGCGGCCGCTCAGGACGGCTTCTTCAGCAGCTCC-3'

[0070] The template is imine reductase Sc IR recombinant plasmid pET28a- Sc IR.

[0071] The PCR reaction system was as follows: 0.5 ng template, 1.5 μl of each pair of mutation primers (10 μM), 8.75 μl MnCl2 (1 mM), 2 × Taq mix 25 μl, and sterile distilled water to 50 μl.

[0072] The error-prone PCR amplification procedure was as follows: (1) denaturation at 95°C for 3 min; (2) denaturation at 95°C for 30 s, (3) annealing at 55°C for 30 s, and (4) extension at 72°C for 1 min. Steps (2) to (4) were repeated for 30 cycles, with a final extension at 72°C for 10 min. The product was stored at 12°C.

[0073] After purification of the PCR amplified product, restriction enzymes Bam HI and Hind III. The PCR amplification product and the vector plasmid pET28a were double-digested at 37 °C for 12 h, the digested products were recovered, and ligated with T4 DNA ligase at 16 °C overnight, and then transformed E. coli BL21 (DE3) competent cells were evenly spread on LB agar 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 deep-well plates. Each well of the primary plate contained 300 μL LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm overnight. 50 μL of the primary seed solution was transferred to each well of the secondary plate containing 600 μL LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm for 3 h until the OD 600At approximately 1.0, IPTG was added to induce the cells at a final concentration of 0.2 mM. The cells were cultured at 16°C for 24 h, centrifuged at 3,500 rpm for 10 min, and the supernatant discarded. 200 μL of lysis buffer (containing 750 mg / L lysozyme and 12 mg / L DNase) was added to each well, shaken to fully resuspend the cells, incubated at 37°C for 1 h, and centrifuged again at 3,500 rpm for 10 min. 50 μL of the supernatant was pipetted into each well of a 96-well microtiter plate. Potassium phosphate buffer (100 mM, pH 7.0) containing 10 mM 2-(2,5-difluorophenyl)pyrroline and 0.15 mM NADPH was added. Activity screening of the mutant library was performed using a microplate reader. Mutants with enhanced activity against the substrate 2-(2,5-difluorophenyl)pyrroline were identified and sequenced. A total of 11 rounds of random mutagenesis were performed.

[0074] Example 2 Imine Reductase Sc Expression, purification and enzyme activity assay of IR mutants

[0075] Pick the recombinant expression single colony of the imine reductase mutant growing on the plate and inoculate it into a 4 mL LB tube containing Kan (50 ng / μL), place it in a 37°C shaker at 200 rpm and culture it for 12-16 h, then transfer the bacterial liquid to 100 mL LB medium containing 50 ng / μL Kan at an inoculation ratio of 1:50, place it in a 37°C shaker at 200 rpm and culture it for 3 h, and wait until its OD 600 When the p-value reaches 0.6-0.8, add 20 μL of IPTG (1 M) to induce expression of the target protein. After culturing at 25°C and 180 rpm for 24 hours, the cells are harvested by centrifugation. The resulting resting cells are suspended in KPB buffer (100 mM, pH 7.0), disrupted by sonication in an ice-water bath, and the supernatant is collected by centrifugation to obtain the cell lysate of the recombinant imine reductase mutant. The cell lysate is freeze-dried to obtain crude enzyme powder.

[0076] The imine reductase mutant protein was purified using Ni column affinity chromatography. The specific method is as follows:

[0077] (1) Equilibrate the Ni affinity column (bed volume 5 ml) with sodium phosphate buffer (20 mM, pH 7.5, containing 500 mM NaCl);

[0078] (2) The resting cells were resuspended in sodium phosphate buffer (20 mM, pH 7.5, containing 500 mM NaCl) and disrupted by ultrasound at 10,000 × gAfter centrifugation for 45 min, the supernatant was collected and passed through a Ni column at a flow rate of 1 mL / min to allow the target protein to bind to the Ni column;

[0079] (3) Elute the impurities that have no binding ability to the Ni column with sodium phosphate buffer (20 mM, pH 7.5, containing 500 mM NaCl) containing 0-50 mM imidazole;

[0080] (4) Elute the target protein with sodium phosphate buffer (20 mM, pH 7.5, containing 500 mM NaCl) containing 250 mM imidazole;

[0081] (5) The collected protein samples were subjected to SDS-PAGE electrophoresis. 20 μl of sample was added to 5 μl of 5× SDS loading buffer and heated. The sample volume for electrophoresis was 10 μl. Protein Molecular Weight Marker was purchased from Thermo Fisher Scientific. The concentration of the SDS-PAGE gel was 12%. The voltage was 90 V for concentration and then changed to 120 V for separation.

[0082] The activity of the recombinant imine reductase was determined by measuring the absorbance change at 340 nm using 10 mM 2-(2,5-difluorophenyl)pyrroline and 0.15 mM NADPH in potassium phosphate buffer (100 mM, pH 7.0) as substrates. The results are shown in Table 1.

[0083] Table 1 Sc Properties of IR and its mutants

[0084]

[0085] in, Sc IR WT Refers to the natural imine reductase with the amino acid sequence shown in SEQ ID No.2 Sc IR.

[0086] Example 3 Sc IR and its mutants Sc Activity of IRM3 towards various 2-substituted pyrrolines

[0087] When using various 2-substituted pyrrolines 1a-1i as active substrates, Sc IR and its mutants Sc IR M3 Activity assays were performed and the results are shown in Table 2. For most substrates, the mutant ScIR M3Compared with the parent, the activities of all mutants were significantly improved. For 1d, 1g, 1h, and 1k, the activities were even improved from zero to high, indicating that the mutants have better universality for 2-arylpyrroline substrates.

[0088] Table 2 Sc IR and its mutants Sc IR M3 Activity towards various 2-substituted pyrrolines

[0089]

[0090] Example 4 Imine reductase mutant catalyzes the asymmetric reduction of 2-(2,5-difluorophenyl)pyrroline

[0091] 5 mL 100 g / L imine reductase mutant ScIR M1 ScIR M2 and ScIR M3 Wet cell disruption solution (KPB 7.0 buffer, 100 mM), BmGDH 20 mg / mL (lyophilized enzyme powder, 5 U / mg), 0.15 mM NADP + The reaction mixture was prepared by mixing 2-5 g / L of the substrate 2-(2,5-difluorophenyl)pyrroline (2-(2,5-difluorophenyl)pyrroline) in 1% DMSO and 1.5 equivalents of glucose to 10 mL with KPB 7.0 buffer. The reaction mixture was incubated at 30°C, with 100 μL of samples taken at intervals throughout the reaction. 20 μL of 10 mM sodium hydroxide solution was added, and 500 μL of ethyl acetate was added for thorough mixing. The mixture was centrifuged at 12,000 rpm for 5 min. The upper organic phase was collected and dried over anhydrous sodium sulfate for 8 h. The reaction was then analyzed by gas chromatography. The results are shown in Table 3. At the same catalyst loading, the wild-type enzyme achieved a conversion of only 26% at a substrate loading of 5 g / L. However, the mutant ScIRM3 converted 80 g / L of substrate within 5 h, demonstrating a significant improvement in the catalytic performance of the mutant compared to the parent enzyme, with a space-time yield of 384 g / L / d.

[0092] Table 3 Sc IR and its mutants catalyze the conversion of the substrate 2-(2,5-difluorophenyl)pyrroline

[0093]

[0094] aThe reaction mixture (10 mL) contained 2-100 g / L substrate, 5 mLcell free extract (from 100 g / L wet

[0095] Example 5 Larotrectinib Chiral Intermediate ( R Enzymatic Scale Synthesis of )-2-(2,5-difluorophenyl)pyrrolidine

[0096] In a 10-L reactor, 9.9 L KPB buffer (100 mM, pH 7.0), 1000 g glucose, 800 g substrate 2-(2,5-difluorophenyl)pyrroline (dissolved in 100 ml DMSO), 0.5 g coenzyme NADP were added. + , 500 g expression recombinant Sc IR M3 The resting cells of 10 kU glucose dehydrogenase crude enzyme powder were reacted at a constant temperature of 30 ° C and mechanical stirring at 150 rpm. After 5 hours, the conversion rate reached 98%. The product obtained by the reaction was extracted with ethyl acetate, added with an appropriate amount of anhydrous sodium sulfate, dried overnight, and the solvent was removed by rotary evaporation to obtain 656 g of product ( R )-2-(2,5-difluorophenyl)pyrrolidine with an isolated yield of 82% and an optical purity of 99%.

[0097] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. Those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An imine reductase mutant, characterized in that: The imine reductase mutant is selected from Sc IR M1 、 Sc IR M2 or Sc IR M3 , in, Sc IR M1 The amino acid sequence is as follows: 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 tryptophan; Sc IR M2 The amino acid sequence is as follows: the valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 is replaced by cysteine, the phenylalanine at position 177 is replaced by tryptophan, the methionine at position 169 is replaced by tryptophan, the tryptophan at position 185 is replaced by leucine, the serine at position 211 is replaced by glycine, the methionine at position 270 is replaced by glutamic acid, the glycine at position 214 is replaced by phenylalanine, and the valine at position 232 is replaced by alanine; Sc IR M3 The amino acid sequence is as follows: the valine at position 122 of the amino acid sequence shown in SEQ ID No. 2 is replaced by cysteine, the phenylalanine at position 177 is replaced by tryptophan, the methionine at position 169 is replaced by tryptophan, the tryptophan at position 185 is replaced by leucine, the serine at position 211 is replaced by glycine, the methionine at position 270 is replaced by glutamic acid, the glycine at position 214 is replaced by phenylalanine, the valine at position 232 is replaced by alanine, the glutamic acid at position 45 is replaced by alanine, the aspartic acid at position 53 is replaced by threonine, the phenylalanine at position 265 is replaced by leucine, and the serine at position 38 is replaced by aspartic acid.

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

3. A recombinant expression vector comprising the nucleic acid according to claim 2. A recombinant expression transformant comprising the recombinant expression vector according to claim 3 .

5. A method for preparing the imine reductase mutant according to claim 1, characterized in that: The recombinant expression transformed bacteria according to claim 4 are cultured to obtain an imine reductase mutant from the culture.

6. The imine reductase mutant according to claim 1 or the resting cells comprising the recombinant expression transformed bacteria according to claim 4 catalyzes the asymmetric reduction of 2-(2,5-difluorophenyl)pyrroline to synthesize the chiral intermediate of larotrectinib ( R )-2-(2,5-difluorophenyl)pyrrolidine.

7. The use according to claim 6, characterized in that: Using 2-(2,5-difluorophenyl)pyrroline as a substrate and in the presence of coenzyme NADPH, the imine reductase mutant according to claim 1 or the resting cells comprising the recombinant expression transformant according to claim 4 are used to catalyze the asymmetric reduction of 2-(2,5-difluorophenyl)pyrroline to prepare the chiral intermediate of larotrectinib ( R )-2-(2,5-difluorophenyl)pyrrolidine, and NADPH is oxidized to NADP + .

8. The imine reductase mutant according to claim 1 Sc IR M3 Application of the invention in catalyzing the asymmetric reduction of 2-arylpyrroline derivatives to synthesize chiral amines, characterized in that: The 2-aryl pyrroline derivative is selected from one of the following compounds: 。

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