Carbonyl reductase mutant and its application in the synthesis of ibrutinib key intermediate

Abstract

The present invention relates to a carbonyl reductase mutant and its application in the synthesis of the key intermediate (S)-N-Boc-3-hydroxypiperidine of ibrutinib. It mainly relates to a mutant with significantly improved thermal stability of carbonyl reductase, its coding nucleic acid, recombinant expression vector, recombinant expression transformant, and the application of using the carbonyl reductase mutant or carbonyl reductase catalyst to catalyze the asymmetric reduction of N-Boc-3-piperidone to produce the key intermediate (S)-N-Boc-3-hydroxypiperidine of ibrutinib. Compared with the prior art, the carbonyl reductase mutant of the present invention has advantages such as high catalytic activity and good thermal stability, and has good industrial application prospects in the preparation of drug intermediates such as ibrutinib.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a carbonyl reductase mutant and its application in the synthesis of (S)-N-Boc-3-hydroxypiperidine, a key intermediate of Ibrutinib. Background Art

[0002] Many bioactive molecules and drugs have a piperidine ring structure. As a key precursor for the synthesis of many drugs, chiral hydroxypiperidine has attracted increasing interest in the pharmaceutical industry. Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, was approved by the US Food and Drug Administration in 2013 for the treatment of specific lymphoma and leukemia cancers. Due to its good safety and strong activity, Ibrutinib ranked third in the global small molecule drug sales in 2019. And (S)-N-Boc-3-hydroxypiperidine ((S)-NBHP), as a key pharmaceutical intermediate and chiral source for the synthesis of Ibrutinib, has an expanding market.

[0003] The preparation methods of chiral N-Boc-3-hydroxypiperidine mainly include chemical methods and biotransformation methods. In the diastereoisomer separation method of chemical methods, the racemic piperidinol is dissolved in a chiral acid of a single configuration, and the piperidinol of a specific configuration forms a salt with the chiral acid and precipitates, so as to achieve the separation from the piperidinol of the other configuration. This method has low resolution efficiency and high cost; the chemical asymmetric synthesis method uses a metal catalyst, which has low catalytic efficiency and harsh reaction conditions. The asymmetric reduction of a ketone substrate mediated by a biocatalyst to produce a chiral alcohol is a more environmentally friendly and sustainable process, with the advantages of high stereoselectivity, moderate reaction conditions, no need for expensive co-substrates, and simple reaction steps.

[0004] In early studies, Romain Lacheretz et al. used carrot tissues as biocatalysts to reduce N-Boc-3-piperidone, obtaining (S)-NBHP with an ee value of 95% and a low yield (73%) (Organic Letters, 2009, 11: 1245-1248). Ju et al. achieved the enzymatic synthesis of (S)-NBHP on a gram scale (100 g / L, 99% ee) through a fed-batch strategy using the ketoreductase KRED (Organic Process Research & Development, 2014, 18: 827-830). Chen et al. found that a thermostable ketoreductase AKR showed good industrial value for the preparation of (S)-NBHP (200 g / L, 99% ee), but it took 16 h to obtain a 99% conversion rate (Applied biochemistry and Biotechnology, 2017, 181: 1304-1313). Xu et al. co-expressed alcohol dehydrogenase TbADH and glucose dehydrogenase, which could catalyze the conversion of 100 g / L of N-Boc-3-piperidone at a wet cell mass of 50 g / L, with a conversion rate of 96%, but the catalyst dosage for this reaction was relatively large (RSC Advances, 2019, 9: 2325-2331). Wei et al. found that FsADH had high catalytic activity towards N-Boc-3-piperidone and could catalyze 597 g / L of the substrate to obtain a 99% conversion rate and 99% ee of (S)-NBHP after 24 h of reaction, but the final product yield was low (58%) (Biochemical Engineering Journal, 2022, 178, 108300). In the catalytic reaction of YGL039W, a substrate load of up to 400 g / L could be tolerated, but the stereoselectivity would be reversed (Catalysis Communications, 2017, 97: 5-9).

[0005] As biocatalysts, enzymes play a key role in the bioeconomy, and their application scope is constantly expanding, including the sustainable and green production of fine chemicals and biofuels. However, compared with traditional chemical catalysts, the application of enzymes is easily limited by poor stability. Most natural enzymes can only catalyze reactions under mild conditions. Excessive reaction temperature easily leads to changes in the structural function of enzymes, thereby affecting reaction performance, with a large amount of enzyme used and a low reaction conversion rate. Therefore, exploring reductases with excellent catalytic activity, thermal stability, strict stereoselectivity, and high substrate / product tolerance is crucial for the industrial biocatalytic asymmetric synthesis of (S)-NBHP. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a carbonyl reductase mutant and its application in the synthesis of the key intermediate of ibrutinib through protein engineering modification.

[0007] Through rational design of the protein structure, the present invention provides several carbonyl reductase mutants with significantly improved thermal stability, solves the problem that their poor thermal stability cannot be practically applied, and expands their practical application value in the synthesis of chiral alcohols.

[0008] Specifically, the present invention provides a carbonyl reductase mutant with high catalytic activity and significantly improved thermal stability, its gene, a recombinant expression vector containing the gene, and a recombinant expression transformant, and uses the recombinant carbonyl reductase as a catalyst to efficiently catalyze the asymmetric reduction of N-Boc-3-piperidone.

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

[0010] One of the technical solutions of the present invention: Provide a carbonyl reductase mutant with significantly improved thermal stability and maintaining high catalytic activity. The carbonyl reductase mutant is a derivative protein with improved thermal stability formed by substituting one or several amino acids in the amino acid sequence shown in SEQ ID No.2, and select the proteins corresponding to the following amino acid sequences:

[0011] (1) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No.2 with glutamine;

[0012] (2) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No.2 with glutamine, and replace the histidine at position 276 with leucine;

[0013] (3) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No.2 with glutamine, and replace the histidine at position 276 with cysteine;

[0014] (4) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No.2 with glutamine, replace the histidine at position 276 with leucine, and replace the glutamine at position 30 with leucine;

[0015] (5) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No.2 with glutamine, replace the histidine at position 276 with cysteine, and replace the glutamine at position 30 with methionine;

[0016] (6) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and alanine at position 313 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine, respectively;

[0017] (7) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and lysine at position 334 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine, respectively;

[0018] (8) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and alanine at position 302 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine, respectively;

[0019] (9) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and lysine at position 48 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and cysteine, respectively;

[0020] (10) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313 and alanine at position 302 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine and arginine, respectively;

[0021] (11) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313 and threonine at position 96 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine and proline, respectively;

[0022] (12) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313 and lysine at position 334 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine and arginine, respectively.

[0023] Based on the carbonyl reductase CgKR1-F92C / F94W in the laboratory enzyme library, the present invention uses a rational design strategy of a computer to modify the thermal stability of CgKR1-F92C / F94W, and obtains a carbonyl reductase mutant with significantly improved thermal stability and high catalytic activity towards N-Boc-3-piperidone. Among them, the amino acid sequence of the carbonyl reductase CgKR1-F92C / F94W is shown in SEQ ID No.2, and it can effectively catalyze the asymmetric reduction of N-Boc-3-piperidone to obtain the target product (S)-N-Boc-3-hydroxypiperidine.

[0024] Among them, the carbonyl reductase CgKR1-F92C / F94W has been reported in the article "Preparation of Structurally Diverse Chiral Alcohols by Engineering Ketoreductase CgKR1" (ACS Catal. 2017, DOI: 10.1021 / acscatal.7b01933).

[0025] In one embodiment of the present invention, the carbonyl reductase CgKR1-F92C / F94W is derived from Candida glabrata.

[0026] The second technical solution of the present invention: provides an isolated nucleic acid, and the nucleic acid is a nucleic acid molecule encoding the carbonyl reductase mutant.

[0027] The third technical solution of the present invention: provides a recombinant expression vector containing the nucleic acid of the carbonyl reductase mutant.

[0028] The recombinant expression vector is obtained by cloning the nucleic acid of the carbonyl reductase mutant into various expression vectors by conventional methods in the art. The expression vectors include various conventional vectors in the art, such as commercially available plasmids, phages or viral vectors, etc., and preferably the plasmid pET-28a(+).

[0029] The fourth technical solution of the present invention: also provides a recombinant expression transformant containing the gene of the carbonyl reductase mutant.

[0030] The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into a host cell. The host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant expression vector can stably replicate itself, and the gene of the carbonyl reductase mutant of the present invention carried by it can be effectively expressed. The host cell is preferably Escherichia coli, and more preferably Escherichia coli E. coli BL21(DE3). Transforming the recombinant expression vector into Escherichia coli E. coli BL21(DE3) can obtain the preferred recombinant expression transformant of the present invention. The transformation method therein is a conventional method in the art, such as heat shock method, electroporation method, etc., and more preferably the heat shock method.

[0031] Technical solution five of the present invention: Provide a preparation method of the recombinant carbonyl reductase or its mutant.

[0032] The preferred preparation method of the recombinant carbonyl reductase or its mutant of the present invention is: Cultivate the recombinant expression transformant as described above, and isolate the recombinantly expressed carbonyl reductase. The medium used for cultivating the recombinant expression transformant is any medium in the art that can enable the transformant to grow and produce the recombinant carbonyl reductase of the present invention. The medium is preferably LB medium, and its formula is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. The culture method and culture conditions, etc. can be appropriately selected according to factors such as the type of host cell and the culture method, as long as the transformant can grow and produce the recombinant carbonyl reductase.

[0033] The specific operation of cultivating the recombinant expression transformant can be carried out according to the conventional operation in the art. Preferably, the recombinant Escherichia coli of the present invention is inoculated into LB medium containing kanamycin and cultured at 37 °C. When the OD 600 of the culture solution reaches 0.5 - 1.0, add β-D-isopropyl-thiogalactopyranoside (IPTG) with a final concentration of 0.1 - 0.5 mmol / L for induction, and continue to culture at 16 °C for 24 h to efficiently express the carbonyl reductase of the present invention. After the culture is completed, centrifuge to collect the precipitated bacterial cells, which are the resting cells of the recombinant expression transformant; suspend the obtained cells in sodium phosphate buffer (PBS, 100 mmol / L, pH 6.0), ultrasonically disrupt, centrifuge the disrupted solution, and collect the supernatant to obtain the crude enzyme solution of the recombinant carbonyl reductase.

[0034] Technical solution six of the present invention: Provide a carbonyl reductase catalyst, which is any one of the following forms:

[0035] (1) Cultivate the recombinant expression transformant, and isolate the transformed cells containing carbonyl reductase;

[0036] (2) Disrupt the transformed somatic cells containing the carbonyl reductase to obtain a cell lysate containing the carbonyl reductase, i.e., a crude enzyme solution;

[0037] (3) Purify the cell lysate containing the carbonyl reductase to obtain a pure enzyme solution.

[0038] The present invention provides a specific activity detection of the enzyme: The activity of the carbonyl reductase is measured using a UV-visible spectrophotometer and calculated by detecting the change in the absorbance value of NADPH at 340 nm. The activity measurement system is 1 mL, including 970 μL of PBS buffer (100 mM, pH 6.0), 10 μL of the substrate N-Boc-3-piperidone (200 mM, solubilized with ethanol), 10 μL of NADPH (17.5 mM), and 10 μL of the enzyme solution (diluted to an appropriate concentration). All substances in the system are sequentially added to a cuvette, mixed evenly, and then placed in a UV spectrophotometer to measure the change in absorbance value at 30 °C. The unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute. The enzyme activity calculation formula is as follows:

[0039] Enzyme activity (U) = EW × V × 10 3 / (6220 × l)

[0040] In the formula, EW is the change in absorbance at 340 nm within 1 minute; V is the volume of the reaction solution, with the unit of mL; 6220 is the molar extinction coefficient of NADPH, with the unit of L / (mol·cm); l is the optical path length, with the unit of cm. 1 unit of enzyme activity (U) corresponds to the amount of enzyme required to catalyze the oxidation of 1 μmol of NADPH per minute under the above conditions.

[0041] Technical solution seven adopted in the present invention: A method for asymmetric reduction of N-Boc-3-piperidone to synthesize (S)-N-Boc-3-hydroxypiperidine using the recombinant carbonyl reductase mutant or carbonyl reductase catalyst as described above.

[0042] The structure of the N-Boc-carbonyl azacyclic ring is as follows:

[0043]

[0044] Using N-Boc-piperidone as the substrate for asymmetric reduction to synthesize (S)-N-Boc-3-hydroxypiperidine, coupling carbonyl reductase CgKR1 with glucose dehydrogenase BmGDH to achieve the coenzyme cycle of NADPH, as shown schematically below:

[0045]

[0046] The conditions for the asymmetric reduction reaction can be selected according to the conventional conditions of such reactions in the art. Preferably, the application includes the following steps: adding Escherichia coli whole cells expressing recombinant carbonyl reductase and glucose dehydrogenase into a reaction buffer, adding a substrate, glucose, and NADP + , and mixing and reacting at a certain temperature. The dosage ratio of the engineered carbonyl reductase to N-Boc-3-piperidone is preferably 80 kU / mol to 240 kU / mol of the substrate, and the preferred dosage ratio of glucose to N-Boc-3-piperidone is 200 g / mol to 300 g / mol. The reaction buffer is a conventional buffer in the laboratory, with a pH range of 5.5 to 7.0, preferably sodium phosphate buffer, and the preferred concentration is 0.1 to 0.2 mol / L. The dosage of the additionally added NADP + is preferably 0 to 0.1 mmol / L. The reaction temperature is preferably 35 to 45 °C, and the reaction process is preferably carried out under stirring conditions. During the reaction process, samples are taken intermittently to measure the reaction conversion rate, and the reaction time is based on the time when the substrate is completely converted or the reaction conversion rate stops increasing, generally 1 to 24 h. The reaction conversion rate is analyzed by gas chromatography.

[0047] After the asymmetric reaction is completed, the reaction solution is extracted with an equal volume of a conventional water-insoluble organic solvent in the art, such as ethyl acetate, butyl acetate, dichloromethane, or methyl tert-butyl ether, and then dried with anhydrous magnesium sulfate.

[0048] Among them, (S)-N-Boc-3-hydroxypiperidine is a key intermediate for the synthesis of the drug ibrutinib.

[0049] Compared with the prior art, the innovative and improved effects of the present invention are as follows:

[0050] The engineered carbonyl reductase mutant of the present invention has the advantages of high catalytic activity, significantly improved thermal stability, and good selectivity. The optimal mutant can convert 100 g / L of the substrate within 4 h, with a conversion rate reaching 99%, and all the obtained products are in the S-configuration and the optical purity is greater than 99% ee, and the enzyme loading is reduced to 5 g / L. Therefore, when the carbonyl reductase of the present invention catalyzes the reduction of N-Boc-3-piperidone, it not only has a small amount of enzyme used, high optical purity, but also a short reaction time, and has good application prospects in the production of drug intermediates such as ibrutinib. Detailed Embodiments

[0051] The present invention will be described in detail below with reference to specific embodiments.

[0052] Unless otherwise specified, the specific experiments in the following examples are carried out according to the conventional methods and conditions in the art, or in accordance with the product specifications.

[0053] The sources of the materials in the following examples are:

[0054] The recombinant plasmid CgKR1-F92C / F94W was constructed in the early stage of the laboratory (those skilled in the art can prepare it by conventional means in the field of biotechnology according to the sequence of CgKR1-F92C / F94W). Competent cells of Escherichia coli E. coli BL21(DE3) and 2×Prime Star were both purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0055] Example 1 Construction of carbonyl reductase CgKR1-F92C / F94W and its mutant plasmid

[0056] Using the recombinant plasmid CgKR1-F92C / F94W as a template, upstream and downstream primers were designed for the sites to be mutated.

[0057] Table 1. Primers used for constructing mutants with CgKR1-F92C / F94W as the starting parent

[0058]

[0059] The gene sequence of carbonyl reductase CgKR1-F92C / F94W is shown in SEQ ID No.1.

[0060] The PCR system was as follows: 10 μL of PrimeSTAR(HS), 6 μL of ddH2O, 1 μL of DMSO, 1 μL each of the upstream primer and the downstream primer, and 1 μL of the template plasmid. The PCR amplification program was: pre-denaturation at 98°C for 3 min, followed by 15 cycles as follows: denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 7 min, and finally incubation at 72°C for 10 min. PCR product digestion: After the PCR amplification was completed, 2 μL of DPnⅠ and 2 μL of Cutsmart were added and placed in a 37°C incubator for 2 - 3 h to obtain the mutant plasmid.

[0061] Example 2 Preparation of recombinant expression transformants of mutant carbonyl reductase

[0062] The mutant plasmid amplified in Example 1 was transformed into Escherichia coli E. coli BL21, and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET28a-CgKR1-F92C / F94WM1-M12.

[0063] Example 3 Preparation of carbonyl reductase CgKR1-F92C / F94W and its mutants

[0064] The recombinant expression transformant obtained in Example 2 was inoculated into an LB test tube containing kanamycin at a final concentration of 50 μg / mL, and cultured in a shaker at 37°C for 8 - 12 h. Then, the bacterial solution was added to a TB shake flask containing kanamycin at a final concentration of 50 μg / mL at an inoculation amount of 1% (v / v), and cultured in a shaker at 37°C for about 3 h until the OD 600 reached 0.6 - 0.8. Subsequently, IPTG (final concentration of 0.2 mM) was added, and the culture was continued in a shaker at 16°C for about 20 - 24 h. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, washed with physiological saline to obtain resting cells, and resuspended in 10 mL of PBS buffer (100 mM, pH 6.0) to obtain mutant wet cells.

[0065] The cell suspension was sonicated: at a power of 400 W, working for 2 s and intermittent for 3 s, for a total of 15 min. The lysate was centrifuged (4°C, 12000 rpm, 30 min), and the supernatant was taken. At this time, the supernatant was the crude enzyme solution. The crude enzyme solution was purified using a nickel column to obtain the mutant pure enzyme.

[0066] The amino acid sequence of the carbonyl reductase mutant is one of the following sequences:

[0067] (1) Aspartic acid at position 138 of the amino acid sequence shown in SEQ ID No.2 was replaced with glutamine, and this mutant was named M1;

[0068] (2) Aspartic acid at position 138 of the amino acid sequence shown in SEQ ID No.2 was replaced with glutamine, histidine at position 276 was replaced with leucine, and this mutant was named M2;

[0069] (3) Aspartic acid at position 138 of the amino acid sequence shown in SEQ ID No.2 was replaced with glutamine, histidine at position 276 was replaced with cysteine, and this mutant was named M3;

[0070] (4) Aspartic acid at position 138 of the amino acid sequence shown in SEQ ID No.2 was replaced with glutamine, histidine at position 276 was replaced with leucine, and glutamine at position 30 was replaced with leucine, and this mutant was named M4;

[0071] (5) Aspartic acid at position 138 of the amino acid sequence shown in SEQ ID No.2 was replaced with glutamine, histidine at position 276 was replaced with cysteine, and glutamine at position 30 was replaced with methionine, and this mutant was named M5;

[0072] (6) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and alanine at position 313 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine respectively, and name this mutant M6;

[0073] (7) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and lysine at position 334 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine respectively, and name this mutant M7;

[0074] (8) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and alanine at position 302 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and arginine respectively, and name this mutant M8;

[0075] (9) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, and lysine at position 48 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, and cysteine respectively, and name this mutant M9;

[0076] (10) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313, and alanine at position 302 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine, and arginine respectively, and name this mutant M10;

[0077] (11) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313, and threonine at position 96 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine, and proline respectively, and name this mutant M11;

[0078] (12) Replace aspartic acid at position 138, histidine at position 276, glutamine at position 30, alanine at position 313, and lysine at position 334 in the amino acid sequence shown in SEQ ID No.2 with glutamine, cysteine, methionine, arginine, and arginine respectively, and name this mutant M12.

[0079] Example 4 Determination of the half-life of recombinant carbonyl reductase CgKR1-F92C / F94W and its mutants

[0080] The pure enzyme obtained in Example 3 was diluted with PBS buffer (100 mM, pH 6.0), incubated in a water bath at 50 °C, and a certain amount of enzyme solution was taken out at intervals, left to stand on ice for 5 min, and then its activity was measured. The half-life was calculated by the first-order inactivation equation. The first-order inactivation equation is ln(V / V0) = -k D t, where the half-life (t 1 / 2 ) = 0.693 / k D . k D is the inactivation rate constant; V is the residual activity, and V0 is the initial activity.

[0081] Table 2 provides a list of the carbonyl reductase CgKR1-F92C / F94W mutants with relevant activities and improved stability in the present invention. In the list of stability (specifically, the half-life of the mutants at 50 °C), compared with the parent CgKR1-F92C / F94W, one plus sign "+" indicates that the stability of the mutant protein is increased by 1-10 times; two plus signs "++" indicate that the stability of the mutant protein is increased by 10-50 times; three plus signs "+++" indicate that the stability of the mutant protein is increased by 50-100 times.

[0082] Table 2. Carbonyl reductase CgKR1-F92C / F94W M1-M12 mutants with improved thermal stability

[0083]

[0084] Examples 5-6 Reduction of N-Boc-3-piperidone by the carbonyl reductase parent M0 and mutant M12

[0085] A 10 mL reaction was carried out using a magnetic stirrer at 40 °C. The reaction mixture contained 0.996 g of the substrate N-Boc-piperidone (500 mM), 1.35 g of glucose (750 mM), 0.787 mg of NADP + (0.1 mM), 0.05 g of the wet E. coli cells of the parent or mutant (5 g / L), 0.166 g of the lyophilized BmGDH enzyme powder (500 U), 9.5 mL of PBS buffer (100 mM, pH 6.0), and 0.5 mL of ethanol as a substrate co-solvent. During the reaction, the pH of the reaction was controlled by dropping 2 M Na2CO3 to keep it around 6.0. At the same time, samples were taken regularly during the reaction until the reaction ended. Each sample was 100 μL, and 500 μL of ethyl acetate was added for shaking extraction. After centrifugation, the supernatant was taken and dried with anhydrous MgSO4. The conversion rate was calculated by gas chromatography analysis. The products obtained were all in the S-configuration with an optical purity greater than 99% ee.

[0086] Table 3. Results of the reduction reaction of N-Boc-3-piperidone catalyzed by carbonyl reductase and mutants

[0087]

[0088]

[0089] For the other mutants M1-M11, the conversion rates of their catalytic reduction reactions of N-Boc-3-piperidone are all above 90%.

[0090] The sequence listing involved in the present invention is as follows:

[0091] SEQ ID NO.1

[0092]

[0093] SEQ ID NO.2

[0094] MASDNSNTTVFVSGATGFIAQHVVRQLLDQNYKVIGSVRSAEKGDHLKNVIFKGGDFNYEIVKDISDPTAFDHVFEKHGKDIKVVLHTASPCHWNTTDIEKDLLIPAVNGTKGILESIKKYAAQTVERVVVTSSFAADSSTVDMFYAKDSSKTITEESWNQDTWESCQSDPIRGYCGSKKFAEKAAWDFYNANKDSVKFKLSIINPVYVFGPQNYVEPGKKILNTSSEVINSLVHLKKDDPLPEFAGGHIDVRDVAKAHILAFQKDELIEQRLMLHAGLFTTQTLLDIINEQFPELKGKIPAGKPGTGNPDDALTPVDNSKTKKLLGFEFIDLKKDLYDTISQILEAEKNSN

[0095] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that 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 efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that, Select the protein corresponding to the following amino acid sequences: (1) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine; (2) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine and replace the histidine at position 276 with leucine; (3) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine and replace the histidine at position 276 with cysteine; (4) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with leucine, and replace the glutamine at position 30 with leucine; (5) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, and replace the glutamine at position 30 with methionine; (6) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, and replace the alanine at position 313 with arginine; (7) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, and replace the lysine at position 334 with arginine; (8) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, and replace the alanine at position 302 with arginine; (9) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, and replace the lysine at position 48 with cysteine; (10) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, replace the alanine at position 313 with arginine, and replace the alanine at position 302 with arginine; (11) Replace the aspartic acid at position 138 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, replace the histidine at position 276 with cysteine, replace the glutamine at position 30 with methionine, replace the alanine at position 313 with arginine, and replace the threonine at position 96 with proline; Replace the aspartic acid at position 138, the histidine at position 276, the glutamine at position 30, the alanine at position 313, and the lysine at position 334 in the amino acid sequence shown in SEQ ID No. 2 with glutamine, cysteine, methionine, arginine, and arginine, respectively.

2. An isolated nucleic acid, characterized in that, The nucleic acid is a nucleic acid molecule encoding the carbonyl reductase mutant as claimed in claim 1.

3. A recombinant expression vector comprising the carbonyl reductase mutant nucleic acid as claimed in claim 2.

4. A recombinant expression transformant comprising the carbonyl reductase mutant gene as claimed in claim 2.

5. A carbonyl reductase catalyst, characterized in that, is any one of the following forms: (1) Culturing the recombinant expression transformant as claimed in claim 4 and isolating the transformed cells containing the carbonyl reductase mutant as claimed in claim 1; (2) Disrupting the transformed cells in (1) to obtain a cell lysate containing the carbonyl reductase mutant as claimed in claim 1, i.e., a crude enzyme solution; (3) A purified enzyme solution obtained by purifying the cell lysate containing the carbonyl reductase mutant.

6. The asymmetric reduction synthesis of N -Boc-3-piperidone catalyzed by the recombinant carbonyl reductase mutant according to claim 1 or the carbonyl reductase catalyst according to claim 5 S )- N Application of -Boc-3-hydroxypiperidine; The N -Boc-3-piperidone has the following structure: 。 7. The application according to claim 6, characterized in that, The reaction of asymmetric reduction of N N -Boc-3-piperidone is carried out in the presence of coenzyme NADPH by using the recombinant carbonyl reductase mutant described in claim 1 or the carbonyl reductase catalyst described in claim 5.

8. The application according to claim 7, characterized in that NADPH can be generated through the conversion reaction of glucose and NADP catalyzed by glucose dehydrogenase + ​ 9. The application according to claim 8, wherein The usage ratio of the recombinant carbonyl reductase mutant according to claim 1 or the carbonyl reductase catalyst according to claim 5 to N -Boc-3-piperidone is 80 kU / mol to 240 kU / mol of substrate, and the ratio of glucose to N -Boc-3-piperidone is 200 g / mol to 300 g / mol; the pH range is 5.5 to 7.0, and the reaction temperature is 35 to 45 °C.

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