A carbonyl reductase mutant, and a method for preparing and using the same

Abstract

The application discloses a carbonyl reductase mutant, a preparation method and application thereof, and belongs to the technical field of enzyme engineering. A series of advantageous mutants are obtained by the method of directional evolution in the field of enzyme engineering on the basis of the carbonyl reductase BcKRED from Bacillus cereus. The obtained carbonyl reductase advantageous mutant has high catalytic activity, thermal stability and reversed chiral selectivity compared with the wild-type carbonyl reductase. The application provides a green and efficient way for industrial production of chiral alcohol by using the carbonyl reductase as a biological catalyst, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering, and particularly relates to a carbonyl reductase mutant, a preparation method and use thereof. BACKGROUND

[0002] Enzymes are a class of polypeptide biological catalysts that can accelerate the chemical reactions of living cells (usually by several orders of magnitude). Without enzymes, most biochemical reactions would be too slow to sustain life. Enzymes have high specificity and can maintain their original characteristics during the reaction process. Since the enzyme itself is not consumed in the catalytic process, the enzyme as a catalyst in a specific chemical reaction can significantly reduce the cost.

[0003] Ketoreductase (KRED), also known as carbonyl reductase, can catalyze the reversible oxidation-reduction reaction between aldehyde / ketone and alcohol using NAD(H) or NADP(H) as a hydrogen carrier. KRED plays an important role in biocatalysis, especially in the asymmetric synthesis of chiral drug intermediates. Due to its high stereoselectivity, mild reaction conditions and environmental friendliness, KRED is widely used in the preparation of various key intermediates of drugs (Nature, 2012, 485: 185-194). For example, KRED is used to synthesize important chiral intermediates of various anticancer drugs, cardiovascular drugs and antiparasitic drugs (ibrutinib, benidipine, orixidine); clopidogrel intermediates; statins (such as rosuvastatin, atorvastatin). The traditional chemical synthesis method of these drugs has problems such as high cost, many by-products, and great environmental pressure. Enzymatic catalysis has the characteristics of mild reaction conditions, high stereoselectivity, and environmental friendliness, and is increasingly used in industrial production.

[0004] Enzymes are usually most active under mild conditions (such as room temperature and neutral pH), but industrial environments may have high temperatures, extreme pH, or higher organic solvents (which can cause enzyme inactivation). These reaction conditions limit the use of enzymes as catalysts in industrial production. In addition, the activity and stability of wild-type enzymes are low, and the narrow substrate range and product inhibition also limit their application. Therefore, it is of great application value to explore new ketoreductase mutants with high catalytic activity, high stability and stereoselectivity. SUMMARY

[0005] In order to solve the above-mentioned problems existing in the prior art, the purpose of the present application is to provide a carbonyl reductase mutant, a preparation method and use thereof.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides a carbonyl reductase mutant, which is obtained by mutating at least one of the following positions: 19th, 25th, 30th, 34th, 48th, 50th, 58th, 66th, 72nd, 80th, 92nd, 94th, 98th, 99th, 102nd, 104th, 105th, 124th, 146th, 149th, 157th, 161st, 193rd, 194th, 199th, 201st, 202nd, 203rd, 205th, 207th, 210th, 211th and 246th, based on a wild-type carbonyl reductase.

[0008] Further, the amino acid sequence of the wild-type carbonyl reductase is shown as SEQ ID NO: 2, and the carbonyl reductase mutant has at least 85% identity with the amino acid sequence shown in SEQ ID NO. 2.

[0009] Further, the mutation comprises at least one of the following mutation modes: the amino acid residue at position 19 is mutated to K, the amino acid residue at position 25 is mutated to N, the amino acid residue at position 30 is mutated to C, the amino acid residue at position 34 is mutated to T, the amino acid residue at position 48 is mutated to A, the amino acid residue at position 50 is mutated to Y, the amino acid residue at position 58 is mutated to S, the amino acid residue at position 66 is mutated to L, the amino acid residue at position 72 is mutated to D, the amino acid residue at position 80 is mutated to D, the amino acid residue at position 92 is mutated to E, the amino acid residue at position 94 is mutated to I, L or V, the amino acid residue at position 98 is mutated to L or W, the amino acid residue at position 99 is mutated to W, the amino acid residue at position 102 is mutated to P, the amino acid residue at position 104 is mutated to E, the amino acid residue at position 105 is mutated to I, the amino acid residue at position 124 is mutated to I, the amino acid residue at position 146 is mutated to T, K, V, M, Q, I, the amino acid residue at position 149 is mutated to H, the amino acid residue at position 157 is mutated to A, the amino acid residue at position 161 is mutated to L, the amino acid residue at position 193 is mutated to L, S, G, H, N, D, R, Y, P, K, Q or A, the amino acid residue at position 194 is mutated to R, P, T, E or K, the amino acid residue at position 199 is mutated to S, the amino acid residue at position 201 is mutated to D, the amino acid residue at position 202 is mutated to Q, D, S, F, N, L, A, Y, H, M, E, T, the amino acid residue at position 203 is mutated to W, D, N, I, V, C, Y, T, M, Q, L, the amino acid residue at position 205 is mutated to V, L or Y, the amino acid residue at position 207 is mutated to W, V, M, F, Y, P, I, L, H, A or D, the amino acid residue at position 210 is mutated to D, E, N, P, S or H, the amino acid residue at position 211 is mutated to Y, the amino acid residue at position 246 is mutated to A, N, S, I or D.

[0010] Further, the mutation comprises at least one of the unit point mutations or the combined mutations shown in Table 1.

[0011] The present application also provides a gene encoding the above-mentioned carbonyl reductase mutant.

[0012] The present application also provides a recombinant expression vector comprising the above-mentioned gene, which is selected from a plasmid, an artificial chromosome, a phage or a viral vector.

[0013] Further, the plasmid is a pET-21a vector or a pGEX6p vector.

[0014] The present application also provides a recombinant expression system comprising the above-mentioned recombinant expression vector.

[0015] Further, the recombinant expression system is recombinant Escherichia coli.

[0016] The present application also provides a method for preparing the above-mentioned carbonyl reductase mutant, comprising the following steps:

[0017] (1) constructing a vector containing a gene encoding the above-mentioned carbonyl reductase mutant;

[0018] (2) transforming the vector into a host bacterium, culturing, adding an inducer, and inducing expression of the carbonyl reductase mutant.

[0019] The present application also provides application of the above-mentioned carbonyl reductase mutant in biocatalytic production of chiral alcohols.

[0020] The present application also provides a method for preparing chiral alcohols, which comprises reaction of the above-mentioned carbonyl reductase mutant, a substrate and a coenzyme regeneration system, so as to obtain chiral alcohols; the substrate is wherein a and b are each independently selected from integers from 0 to 5, and R is C 1-8 alkyl.

[0021] Further, the substrate is The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose, NADP + and glucose dehydrogenase.

[0022] The present application has the following beneficial effects:

[0023] The present application obtains a series of advantageous mutants of carbonyl reductase by the method of directed evolution in the field of enzyme engineering based on carbonyl reductase BcKRED from Bacillus cereus. The advantageous mutants of carbonyl reductase obtained by the present application have high catalytic activity, thermal stability and reversed chiral selectivity compared with wild-type carbonyl reductase. The present application provides a green and efficient way for industrial production of chiral alcohols by using carbonyl reductase as a biocatalyst, and has a broad application prospect.

[0024] The substrate used in the embodiments of the present application is:

[0025] The substrate S1 is The substrate S2 is The substrate S3 is

[0026] The product obtained in the embodiments of the present application is:

[0027] The product P1 is The product P2 is The product P3 is

[0028] In the nucleotide mutation code of the present application, the number is the nucleotide site number of mutation, the letter before the number represents the nucleotide before mutation, and the letter after the number represents the nucleotide after mutation. For example, ATG280-282ATT means that the 280th, 281st and 282nd nucleotides are mutated from ATG to ATT, and so on.

[0029] In the amino acid mutation code of the present application, the number is the amino acid site number of mutation, the letter before the number represents the amino acid before mutation, and the letter after the number represents the amino acid after mutation. For example, M94I means that the 94th amino acid is mutated from M to I, and so on.

[0030] In the amino acid mutation code of the present application, the letters before and after the number are the amino acid abbreviations commonly known in the art. For example, V represents Valine (valine), A represents Alanine (alanine), M represents Methionine (methionine), L represents Leucine (leucine), Q represents Glutamine (glutamine), C represents Cysteine (cysteine), K represents Lysine (lysine), T represents Threonine (threonine), I represents Isoleucine (isoleucine), S represents Serine (serine), H represents Histidine (histidine), E represents Glutamic acid (glutamic acid), P represents Proline (proline), F represents Phenylalanine (phenylalanine), D represents Aspartic acid (aspartic acid), Y represents Tyrosine (tyrosine), G represents Glycine (glycine), and N represents Asparagine (asparagine).

[0031] In the combined mutation code of the present application, "+" means that the mutation before "+" and the mutation after "+" are performed. For example, M94I+M193L means that the 94th amino acid is mutated from M to I and the 193rd amino acid is mutated from M to L; ATG280-282ATT+ATG577-579CTG means that the 280th, 281st and 282nd nucleotides are mutated from ATG to ATT and the 577th, 578th and 579th nucleotides are mutated from ATG to CTG, and so on.

[0032] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0033] The above content of the present application is further explained in detail by way of specific embodiments in the form of examples. However, it should not be understood that the above-mentioned subject matter of the present application is limited to the following examples. Any technology achieved based on the above content of the present application falls within the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Chiral HPLC analysis spectrum of chiral product P1 catalyzed by the carbonyl reductase BcKRED mutant M178 and M193 of the present application.

[0035] Figure 2 Chiral HPLC analysis spectrum of chiral product P2 catalyzed by the carbonyl reductase BcKRED mutant M175 of the present application.

[0036] Figure 3 Chiral HPLC analysis spectrum of chiral product P3 catalyzed by the carbonyl reductase BcKRED mutant M178 of the present application.

[0037] Figure 4 Organic solvent tolerance test of the carbonyl reductase BcKRED mutant M178 and M193 of the present application. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0039] The carbonyl reductase BcKRED involved in the present application is obtained by gene mining from the NCBI database using genome mining technology. The gene (wild type) is from Bacillus cereus, uniprot: Q8RJB2. The related gene is synthesized by a gene synthesis company. The nucleotide sequence of BcKRED is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0040] The carbonyl reductase BcKRED mutant involved in the present application is obtained by mutation on the basis of the wild type BcKRED. The amino acid mutation sites of the carbonyl reductase BcKRED mutant, the corresponding nucleotide mutation sites and the mutation mode are shown in Table 1:

[0041] Table 1 Mutation sites and mutation modes of the carbonyl reductase BcKRED mutant of the present application

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The construction, heterologous expression, purification and freeze-dried powder preparation method of the carbonyl reductase BcKRED recombinant bacteria of the application are as follows:

[0059] The carbonyl reductase BcKRED is constructed on the pET-21a(+) vector with a C-terminal His tag, which is used for the purification of BcKRED wild type and mutants. At the same time, BcKRED is also constructed on the pGEX6p vector, which is used for the construction and screening of mutant library, the preparation of freeze-dried powder and activity determination.

[0060] The carbonyl reductase BcKRED is overexpressed by using the E. coli heterologous expression system. Specifically, the plasmid carrying the carbonyl reductase BcKRED gene is transformed into E. coli BL21(DE3) to construct recombinant bacteria. Single colonies are picked into LB (containing kanamycin 50 μg / mL) medium and cultured overnight at 37°C. With a 1% inoculation amount, the culture is transferred into 80 mL TB (containing kanamycin 50 μg / mL) medium and cultured at 37°C for 3 h. When the OD 600 After adding 0.5 mM IPTG for induction, the culture is continued to be cultured at 25°C for 16 h.

[0061] The preparation of the lyophilized powder of carbonyl reductase BcKRED is a general method in the art. Specifically, the recombinant bacteria are centrifuged for 10 min (6,000 rpm, 4°C), and the bacterial bodies are collected. 1 g of the bacterial bodies is added to 7 mL of PBS phosphate buffer (pH 7.2-7.4, 0.01 M). The cells are broken by an ultrasonic cell disruptor, and the supernatant obtained after centrifugation (12,000 rpm, 4°C) is the crude enzyme solution. The crude enzyme solution is quickly frozen with liquid nitrogen and then dried in a freeze dryer to obtain the lyophilized powder of the crude enzyme solution.

[0062] The purification of carbonyl reductase BcKRED is performed by affinity chromatography. 1-1.5 g of the wet bacterial bodies after induction culture are resuspended in 10 mL of Buffer A (1X PBS buffer), broken by ultrasonic waves (ultrasonic waves for 3 s, interval of 2 s, 5 min), and then centrifuged at 13,000 rpm and 4°C for 20 min. The supernatant is added to the column material equilibrated with Buffer A, and then gently mixed on a rotary apparatus for 30 min. Non-specifically bound proteins are eluted with Buffer A, and then the impurities are rinsed with Buffer A containing 20 mM imidazole. Finally, the target protein is eluted with Buffer A containing 250 mM imidazole, and the purity is identified by electrophoresis. The protein concentration is determined by A 280 nm.

[0063] Example 1: Prediction and screening of mutation sites of the substrate binding pocket

[0064] By analyzing the amino acid sequence and three-dimensional spatial structure of carbonyl reductase BcKRED, potential stability mutation sites are predicted and screened.

[0065] First, the amino acid sequence SEQ ID NO. 2 of the wild-type carbonyl reductase BcKRED is used as a search template to perform a protein sequence similarity search in the NCBI database, and homologous sequences with sequence similarity of 30-80% are selected. Then, multiple sequence alignment is performed by ClusterW. According to the results of sequence alignment, the amino acids of the substrate binding pocket are analyzed, and the amino acids with high frequency are used as the first round of predicted mutation sites. The selected mutations are M94I, M94L, M94V, W151M, M193L, F205Y, and F211Y. Through activity determination, the dominant mutants are combined to obtain mutants with improved activity.

[0066] The above mutants are obtained by site-directed mutagenesis using the recombinant plasmids pET-21a(+)-BcKRED and pGEX6p-BcKRED wild type as templates, and the mutants are transformed into Escherichia coli BL21 (DE3) for induction expression, thereby obtaining the mutant enzymes.

[0067] Example 2: Establishment and screening of BcKRED random mutation library

[0068] The gene of mutant M13 obtained by screening in Example 1 was used as a template to amplify the gene of mutant M13 of BcKRED by error-prone PCR. The PCR product was transformed into E. coli Top10 cells using a chemical method, and single colony bacteria were picked and cultured to construct a random mutation library. The specific cell culture and stability screening method is as follows: the E. coli cells cultured and expressed in a 96-well plate were centrifuged at 4,000 rpm for 20 min at 4°C, and the supernatant was removed. 200 μL of cell lysis system (cell lysis system containing 50 mM, pH 7.5 potassium phosphate buffer, 10 mg / mL lysozyme, 1 μg / mL DNase I, 20 mM MgCl2) was added to the 96-well plate, and incubated at 37°C for 1 h. After centrifugation, the supernatant was aspirated. The supernatant was incubated at 40°C for 1 h, centrifuged to obtain the supernatant, and then incubated with a potassium phosphate buffer containing substrate S1 and 1.5 mM NADPH for 20 min. The absorbance of NADPH was measured at 340 nm, and the wells with higher activity were screened to obtain mutants. The activity of the mutants was determined by freeze-dried powder in the subsequent step.

[0069] Example 3: Establishment and screening of BcKRED saturation mutation library

[0070] In order to further improve the catalytic activity and stability of the enzyme, molecular dynamics simulation was performed on the wild-type carbonyl reductase BcKRED using Schrodinger. By analyzing the results of molecular dynamics simulation, residues with large fluctuations were selected. In addition, according to the results of molecular docking, amino acid sites that interact with the substrate were predicted. Based on the results of molecular docking and molecular dynamics simulation, unit point saturation libraries were constructed for sites R246, N146, M193, Q194, F205, F211, M94, W151, V91, Y148. Double-site saturation libraries were constructed for N207-R210, K202-E203 and E98-K99. Degenerate codon NDT / VHG / TGG was used to design primers for the construction of saturation libraries.

[0071] Firstly, PCR experiments were performed to construct NDT / VHG / TGG saturation library using BcKRED-M9 and BcKRED-M10 as templates. Q194, F205, F211, M94, and W151 were used to construct NDT / VHG / TGG saturation library using BcKRED-M9 as template. M193 was used to construct NDT / VHG / TGG saturation library using BcKRED-M10 as template. The PCR products were transformed into E. coli Top10 cells using chemical method, and ~200 single colonies of each library were picked and cultured. The single colonies were inoculated into 96-well culture plates containing 200 μL of LB medium and cultured at 37°C overnight. 30 μL of seed liquid was inoculated into 96-well deep well plates containing 800 μL of LB medium. When the cells reached OD600 of 0.6-0.8 at 37°C, 0.5 mM IPTG was added, and the protein expression was induced at 25°C overnight. After centrifugation, the supernatant was discarded, 200 μL of reaction solution was added, and the reaction was carried out at 37°C for 16 h. The specific reaction system was as follows: substrate S1 2.1 mg / mL, NADP + 0.4 mg / mL, NADPH regeneration system, 10% DMSO, 50 mM potassium phosphate buffer, pH 7.5. After the reaction was completed, 400 μL of acetonitrile was added to terminate the reaction, and the mixture was shaken and centrifuged. After filtration, HPLC detection was performed. By comparing the enzyme activity of the template carbonyl reductase, mutants with higher activity were screened.

[0072] To further improve the catalytic activity of BcKRED, PCR experiments were performed to construct NDT / VHG / TGG saturation library at positions V91, N146, Y148, R246, and N207-R210 using M57 as template. Further, the beneficial mutant M109 obtained in this round of screening was used as template to construct double-site saturation library at positions K202-E203 and E98-K99. By comparing the enzyme activity of the template carbonyl reductase, mutants with higher activity were screened, and dominant mutants were obtained.

[0073] Through the screening of Examples 1-3, 195 potential dominant mutants were finally obtained.

[0074] Example 4: Activity determination of carbonyl reductase BcKRED mutant

[0075] To evaluate the catalytic activity of the obtained mutants, substrate S1 was selected to evaluate the activity of the 195 mutants obtained.

[0076] The reaction flow of substrate S1 is shown below:

[0077]

[0078] The reaction was conducted in a glass flask with a volume of 500 μL, containing 50 mM substrate S1, 1 mg / mL of BcKRED mutant lyophilized powder, and 1 mM NADP. + The reaction mixture consisted of 100 mM D-glucose, 1 mg / mL NADPH regeneration enzyme powder, 100 mM potassium phosphate buffer (pH 7.0), and 10% v / v DMSO. The reaction solution was placed in a shaker (750 rpm, 30 °C) and reacted for 18 h. After the reaction was complete, an equal volume of acetonitrile was added to terminate the reaction. The supernatant was collected after centrifugation, diluted to a certain concentration, and the conversion rate of product P1 was detected by HPLC. The column used for detection was an InfinityLab Poroshell 120EC-C18 (3.0 x 30 mm, 2.7 μm). The conversion rate was calculated based on the product standard curve. The activity assays of the mutant are shown in Table 2.

[0079] Table 2 Catalytic activity of carbonyl reductase BcKRED mutants

[0080]

[0081]

[0082] The results are shown in Table 2. All 195 mutants obtained by screening in this invention showed better catalytic activity for substrate S1 than wild-type M0.

[0083] Chirality determination of product 1 (i.e., P1) was performed on mutants M178 and M193 (e.g. Figure 1 (As shown). Specifically, after the reaction was completed, the product was extracted three times with 500 μL MTBE (methyl tert-butyl ether), and the MTBE layer was collected to obtain the product alcohol. The wild type was determined to be S-configuration (ee, 97%) by HPLC chiral column analysis. The products obtained by catalyzing substrate S1 with mutants M178 and M193 were all S-configuration, and the ee value was improved (ee, >99%), which was superior to that of wild type M0.

[0084] Example 5: Determination of the activity of carbonyl reductase BcKRED mutant in reducing candidate compounds and the chirality of the products

[0085] To further broaden the substrate profile of BcKRED, in this embodiment, the activity and chiral selection of substrate S2 were determined using the dominant mutant M175; and the activity and chiral selection of substrate S3 were determined using the dominant mutant M178. The specific procedures are as follows:

[0086] (1) Reduction of substrate S2

[0087] The reaction flow of substrate S2 is shown below:

[0088]

[0089] Reaction setup: 500 μL reaction setup in potassium phosphate buffer (pH 7.0, 0.1 M) with 20 mM substrate S1, 5 mg / mL of BcKRED mutant lyophilized powder, 1 mM NADP + regeneration system, 10% v / v DMSO. The reaction was placed in a shaker (750 rpm, 30 °C) for 16-18 h.

[0090] Conversion rate determination: After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction, and the supernatant was taken after centrifugation. After dilution to a certain concentration, the product conversion rate was detected by HPLC. The column used was Kinetex 2.6 μm EVO C18 LCColumn 30 x 2.1 mm), and the conversion rate was calculated according to the product standard curve. The conversion rate of the wild type was 27% by HPLC, and the conversion rate of mutant M175 was 93%.

[0091] Product chirality determination: After the reaction was completed, NaOH was first added to adjust the pH to 11, and 500 μL MTBE (methyl tert-butyl ether) was extracted three times, and the MTBE layer was taken to obtain the product alcohol (product 2, i.e. P2). The wild type was S configuration (ee, 88%) by HPLC chiral column. The results are shown in Figure 2 Table 2, and the product obtained by mutant M175 catalyzing substrate S2 was R configuration (ee, >99%).

[0092] (2) Reducing substrate S3

[0093] The reaction scheme of substrate S3 is shown below:

[0094]

[0095] Reaction setup: 500 μL reaction setup in potassium phosphate buffer (pH 7.0, 0.1 M) with 20 mM substrate S1, 5 mg / mL of BcKRED mutant lyophilized powder, 1 mM NADP + regeneration system, 10% v / v DMSO. The reaction was placed in a shaker (750 rpm, 30 °C) for 16-18 h.

[0096] Conversion rate determination: After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction, and the supernatant was taken after centrifugation. After dilution to a certain concentration, the product conversion rate was detected by HPLC. The column used was Kinetex 2.6 μm EVO C18 (LC Column 30 x 2.1 mm). The conversion rates described in this embodiment were calculated based on the product standard curve. HPLC analysis showed a wild-type conversion rate of 26% and a mutant M178 conversion rate of 61%.

[0097] Product chirality determination: After the reaction was complete, the product was extracted three times with 500 μL MTBE (methyl tert-butyl ether). The MTBE layer was collected to obtain the product alcohol (product 3, i.e., P3). HPLC chiral column analysis showed that the wild-type was S-configuration (ee, 82%). Results are as follows... Figure 3 As shown, the product obtained by catalyzing substrate S3 with mutant M178 is in the R configuration (ee, 91%).

[0098] Example 6: Determination of the thermal stability (Tm) of the carbonyl reductase BcKRED mutant

[0099] The melting temperature (T) of the protein was determined using the CFX96 Real-Time PCR Detection System. m The program involved heating from 25°C to 95°C, with a temperature increase of 0.5°C every 30 seconds. The specific reaction system used was as follows: 25 μL of reaction system contained 0.4 mg / mL of purified mutant protein and 10× fluorescent dye (5000X, SYPRO). TM Orange dye was used, and the buffer was 1xPBS. The Tm values ​​of the mutants are shown in Table 3.

[0100] Table 3. Stability determination of dominant mutants of carbonyl reductase BcKRED

[0101] Number Tm / °C ΔTm / °C Number Tm / °C ΔTm / °C M0 58.5 0 M77 71 12.5 M13 60 1.5 M81 59 0.5 M14 63.5 5 M90 59.5 1 M15 63 4.5 M91 67.5 9 M16 64 5.5 M92 69.5 11 M17 61.5 3 M95 69 10.5 M18 63 4.5 M97 68 9.5 M19 62.5 4 M99 69.5 11 M21 60.5 2 M100 70.5 12 M22 63 4.5 M101 70.5 12 M23 62 3.5 M102 70 11.5 M24 59 0.5 M103 71.5 13 M25 64.5 6 M104 71.5 13 M26 62.5 4 M109 58.5 0 M27 59.5 1 M115 59.5 1 M28 59.5 1 M124 72.5 14 M29 60.5 2 M125 62.5 4 M30 64 5.5 M126 73 14.5 M31 64 5.5 M127 73.5 15 M32 60 1.5 M128 73.5 15 M33 61 2.5 M130 58 -0.5 M34 61 2.5 M131 58.5 0 M52 59.5 1 M132 62 3.5 M57 59 0.5 M133 73.5 15 M60 61.5 3 M134 74 15.5 M61 65.5 7 M135 74 15.5 M62 61 2.5 M136 74.5 16 M63 60 1.5 M167 59.5 1 M64 59.5 1 M171 72 13.5 M65 62.5 4 M178 75 16.5 M69 66.5 8 M179 72 13.5 M70 66 7.5 M180 72.5 14 M72 67 8.5 M187 72.5 14 M73 67 8.5 M188 66.5 8 M74 66.5 8 M189 71 12.5 M75 69 10.5 M190 72 13.5 M76 70.5 12 M193 71.5 13

[0102] The results are shown in Table 3. m The values ​​were all higher than those of wild-type M0, and the T values ​​of mutant M178 were also higher. m It can be raised to a maximum of 75℃.

[0103] Experiments have shown that the melting temperature, i.e. thermal stability, of the carbonyl reductase BcKRED mutant screened in this embodiment is significantly better than that of the wild-type M0.

[0104] Example 7: Organic solvent tolerance of carbonyl reductase BcKRED mutants M178 and M193

[0105] Enzyme activity was measured in the range of DMSO (10-50%).

[0106] Reaction setup: A 500 μL reaction mixture was prepared in potassium phosphate buffer (pH 7.0, 0.1 M), containing 2 mg / mL of wild-type and mutant lyophilized carbonyl reductase powder, 50 mM substrate S1, and 1 mM NADP.+ The reaction system was reacted under 10%, 20%, 30%, 40%, 50% v / v DMSO, respectively, and the NADPH regeneration system was used. The reaction conditions were as follows: 30 DEG C, 750 rpm, 16-18 h.

[0107] In this embodiment, in order to ensure the accuracy of the comparison of the stability data of the wild type and the mutant, the determination of the wild type and the mutant is carried out at the same time. As shown in the results, the activity and stability of the mutants M178 and M193 in different concentrations of DMSO are higher than those of the wild type M0. It is proved that the mutant has good organic solvent resistance. Figure 4

[0108] In summary, the present application obtains a series of advantage mutants by the method of directed evolution in the field of enzyme engineering on the basis of the carbonyl reductase BcKRED from Bacillus cereus. The advantage mutant of the carbonyl reductase obtained by the present application has high catalytic activity, thermal stability and reversed chiral selectivity compared with the wild type carbonyl reductase. The present application provides a green and efficient way for the industrial production of chiral alcohol by using the carbonyl reductase as a biological catalyst, and has a broad application prospect.

[0109] The sequence involved in the present application is as follows:

[0110] The wild type BcKRED has the nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2: artificial nucleotide sequence <747bp>

[0111]

[0112] artificial amino acid sequence <249aa>

[0113] ​

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant was obtained by mutation based on the wild-type carbonyl reductase. The amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO:

2. The mutation is one of the following single point mutations or combination mutations:

2. A gene encoding the carbonyl reductase mutant of claim 1.

3. A recombinant expression vector comprising the gene of claim 2, wherein the recombinant expression vector is selected from plasmids, artificial chromosomes, bacteriophages, or viral vectors.

4. The recombinant expression vector according to claim 3, characterized in that, The plasmid is either the pET-21a vector or the pGEX6p vector.

5. A recombinant expression system comprising the recombinant expression vector of claim 3 or 4.

6. The recombinant expression system according to claim 5, characterized in that, The recombinant expression system is recombinant Escherichia coli.

7. A method for preparing the carbonyl reductase mutant of claim 1, characterized in that, The method includes the following steps: (1) Constructing a vector containing a gene encoding the carbonyl reductase mutant of claim 1; (2) The vector was transferred into the host bacteria, cultured, and an inducer was added to induce the expression of carbonyl reductase mutant.

8. The application of the carbonyl reductase mutant of claim 1 in the production of chiral alcohols from bio-enzymatically catalyzed substrates, wherein the substrate is... The chiral alcohol is .

9. A method for preparing chiral alcohols, characterized in that, The method involves reacting the carbonyl reductase mutant, substrate, and coenzyme regeneration system described in claim 1 to obtain a chiral alcohol; the substrate is... .

10. The method according to claim 9, characterized in that, The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose and NADP. + And glucose dehydrogenase.

11. The application of a carbonyl reductase mutant in the production of chiral alcohols from substrates catalyzed by biological enzymes, wherein the carbonyl reductase mutant is obtained by mutation based on wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO:2, and the mutation is: M94L+F205Y+F211Y+A201D+S30C+V161L+Q58S+S102P+T19K+M193K+Q194K+N146K+R246D+N66L+A92E+N207Y+S48A+S80D+T157A+R210W; the substrate is... The chiral alcohol is .

12. A method for preparing chiral alcohols, characterized in that, The method involves the reaction of a carbonyl reductase mutant, a substrate, and a coenzyme regeneration system to obtain a chiral alcohol. The carbonyl reductase mutant is obtained by mutating a wild-type carbonyl reductase. The amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO:

2. The mutation is: M94L+F205Y+F211Y+A201D+S30C+V161L+Q58S+S102P+T19K+M193K+Q194K+N146K+R246D+N66L+A92E+N207Y+S48A+S80D+T157A+R210W. The substrate is... .

13. The method according to claim 12, characterized in that, The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose and NADP. + And glucose dehydrogenase.

14. The application of a carbonyl reductase mutant in the production of chiral alcohols from substrates catalyzed by biological enzymes, wherein the carbonyl reductase mutant is obtained by mutation based on wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO:2, and the mutation is: M94L+F205Y+F211Y+A201D+S30C+V161L+Q58S+S102P+T19K+M193K+Q194K+N146K+R246D+N66L+A92E+N207Y+R210D+S48A+S80D+T157A+K202H+E203L; the substrate is... The chiral alcohol is .

15. A method for preparing chiral alcohols, characterized in that, The method involves the reaction of a carbonyl reductase mutant, a substrate, and a coenzyme regeneration system to obtain a chiral alcohol. The carbonyl reductase mutant is obtained by mutating a wild-type carbonyl reductase. The amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO:

2. The mutation is: M94L+F205Y+F211Y+A201D+S30C+V161L+Q58S+S102P+T19K+M193K+Q194K+N146K+R246D+N66L+A92E+N207Y+R210D+S48A+S80D+T157A+K202H+E203L. The substrate is... .

16. The method according to claim 15, characterized in that, The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose and NADP. + And glucose dehydrogenase.

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