A carbonyl reductase mutant, and a method of 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 genetic engineering and enzyme engineering. The wild-type carbonyl reductase of Stachybotrys chlorohalonata is used as an evolution template, and multiple mutants with catalytic activity, thermal stability and substrate spectrum obviously superior to the wild type are obtained through enzyme engineering technology. The carbonyl reductase mutant can catalyze a plurality of prochiral carbonyl compounds to obtain chiral alcohol with high stereoselectivity (ee>99%). The carbonyl reductase mutant provides a new, efficient and environmentally-friendly technical route for industrialized production of chiral alcohol products, and has very high industrial application potential.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a carbonyl reductase mutant, its preparation method, and its uses. Background Technology

[0002] Ketoreductase (KRED), also known as carbonyl reductase (EC 1.1.1.184), is a class of biocatalysts that rely on NAD(H) / NADP(H) coenzymes for catalytic reactions. It efficiently catalyzes the asymmetric reduction reactions between aldehydes / ketones and chiral alcohols. Due to its excellent stereoselectivity, mild reaction conditions, and environmentally friendly properties, this enzyme has become a key tool in the synthesis of chiral drug intermediates. Carbonyl reductases are used as catalysts in the synthesis of drugs such as etimibe, montelukast, duloxetine, and atorvastatin. Compared to chemical methods, enzymatic synthesis of chiral alcohols requires only one step to catalyze the conversion of ketones to corresponding alcohols, while chemical methods typically require multiple steps. Therefore, carbonyl reductases are increasingly widely used in industrial production.

[0003] Biocatalysis in industrial production often requires overcoming relatively extreme environments, such as high temperatures, strong acids and alkalis, and organic solvents. Enzymes, as catalysts, typically require relatively mild conditions to catalyze reactions, and the conditions in industrial production easily lead to the inactivation of natural enzymes. Therefore, developing KRED mutants with high thermal stability has become a key research focus in the field of carbonyl reductase modification. Furthermore, in enzyme catalysis, the narrow substrate spectrum of wild-type enzymes, which can only catalyze reactions on specific types of substrates, limits their application in industrial fields.

[0004] Therefore, discovering enzymes with a broad substrate spectrum and improving their thermal stability, catalytic activity, and substrate universality through enzyme engineering technology has important application value in industrial production. Summary of the Invention

[0005] In order to solve the above-mentioned problems in the prior art, the purpose of this invention is to provide a carbonyl reductase mutant, its preparation method and uses.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a carbonyl reductase mutant, which is obtained by mutation of wild-type carbonyl reductase. The wild-type carbonyl reductase is similar to SEQ ID NO. The amino acid sequence shown in NO.2 has at least 90% identity, and the mutated site is at least one of the following sites: position 19, position 27, position 58, position 64, position 69, position 71, position 77, position 81, position 84, position 96, position 98, position 114, position 122, position 124, position 135, position 136, position 158, position 175, position 207, position 216, position 238, position 241, position 242, position 245, position 246, position 265, position 268, position 278, position 283, position 289, position 295, position 331, position 336, and position 337.

[0008] Furthermore, the wild-type carbonyl reductase is related to SEQ ID NO. The amino acid sequence shown in NO.2 has 90%, 95%, 96%, 97%, 98%, or 99% or more homology, and the mutation includes at least one of the following mutation methods: mutating the amino acid residue at position 19 to G, the amino acid residue at position 27 to H, the amino acid residue at position 58 to K, the amino acid residue at position 64 to K, the amino acid residue at position 69 to Q, the amino acid residue at position 71 to P, the amino acid residue at position 77 to G, the amino acid residue at position 81 to E, the amino acid residue at position 84 to K, the amino acid residue at position 96 to V, the amino acid residue at position 98 to F, the amino acid residue at position 114 to L, the amino acid residue at position 122 to K, the amino acid residue at position 124 to P, the amino acid residue at position 135 to A, and the amino acid residue at position 136 to V, S, or C, the amino acid residue at position 15... The amino acid residue at position 8 mutates to V, the amino acid residue at position 175 mutates to I, the amino acid residue at position 207 mutates to A, the amino acid residue at position 216 mutates to P or L, the amino acid residue at position 238 mutates to A, the amino acid residue at position 241 mutates to L, V, or A, the amino acid residue at position 242 mutates to G, the amino acid residue at position 245 mutates to N, C, L, I, V, K, T, or M, and the amino acid residue at position 246 mutates... The mutated amino acid residues are w, D, R, I, M, or T. The mutated amino acid residue at position 265 is D, the mutated amino acid residue at position 268 is N, the mutated amino acid residue at position 278 is F or Y, the mutated amino acid residue at position 283 is I, the mutated amino acid residue at position 289 is K, the mutated amino acid residue at position 295 is T, the mutated amino acid residue at position 331 is E, the mutated amino acid residue at position 336 is R, and the mutated amino acid residue at position 337 is A.

[0009] Furthermore, the amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO.2, and the mutation includes at least one of the following single point mutations or combination mutations:

[0010]

[0011]

[0012]

[0013] The present invention also provides a gene encoding the above-mentioned carbonyl reductase mutant.

[0014] The present invention also provides a recombinant expression vector containing the above-mentioned genes, wherein the recombinant expression vector is selected from plasmids, artificial chromosomes, bacteriophages or viral vectors.

[0015] Furthermore, the plasmid is either the pET-21a vector or the pGEX6p vector.

[0016] The present invention also provides a recombinant expression system comprising the above-mentioned recombinant expression vector.

[0017] Furthermore, the recombinant expression system is recombinant Escherichia coli.

[0018] The present invention also provides a method for preparing the above-mentioned carbonyl reductase mutant, the method comprising the following steps:

[0019] (1) Construct a vector containing the gene encoding the above carbonyl reductase mutant;

[0020] (2) The vector was transferred into the host bacteria, cultured, and an inducer was added to induce the expression of the carbonyl reductase mutant.

[0021] This invention also provides the application of the carbonyl reductase mutant in the catalytic production of chiral alcohols from substrates with asymmetric carbonyl structures, wherein the substrates with asymmetric carbonyl structures are... m and n are each independently selected from 0 or 1; R1 and R2 are different and R1 and R2 are each independently selected from C1-9 alkyl, C1-9 alkoxy, heterocyclic, cycloalkyl, aromatic, heteroaromatic; or, R1 and R2 are each independently selected from substituted C1-9 alkyl, C1-9 alkoxy, heterocyclic, cycloalkyl, aromatic, heteroaromatic, wherein the substituents are each independently selected from halogen, C1-6 alkyl, C1-6 alkoxy, halogen-substituted C1-6 alkyl, halogen-substituted C1-6 alkoxy; or, R1 and R2 are linked to form a ring;

[0022] Furthermore, the substrate with the asymmetric carbonyl structure is Among them, R3, R4, R5, R6, and R7 are each independently selected from H, halogen, C1-6 alkyl, C1-6 alkoxy, halogen-substituted C1-6 alkyl, and halogen-substituted C1-6 alkoxy, and R8 is H, halogen, C1-6 alkyl, halogen-substituted C1-6 alkyl, or... R9 is a C1-6 alkyl or halogen-substituted phenyl group. 10 For halogens, R 11 It is a C1-6 alkyl or benzyl group, and a, b, and c are each independently selected from integers from 1 to 10.

[0023] Furthermore, R3 is H or Cl, R4 is H, F, Cl, Br or CH3, R5 is H, F, Cl, CF3 or methoxy, R6 is H, Cl or F, R7 is H, F, Cl, CH3 or CH2Cl, and R8 is H, CF3, CH2Cl, Br or R9 is CH3 or C6H4Cl, R 10 For Br, R 11 It is a C1-6 alkyl or benzyl group, and a, b, and c are each independently selected from integers from 1 to 5.

[0024] Furthermore, the substrate with the asymmetric carbonyl structure is:

[0025]

[0026] The present invention also provides a method for preparing chiral alcohols, wherein the method comprises reacting the above-mentioned carbonyl reductase mutant, a substrate with an asymmetric carbonyl structure, an organic reagent, a reaction buffer, and a coenzyme regeneration system to obtain the chiral alcohol;

[0027] The concentration of the carbonyl reductase mutant is 0.1-10 mg / mL;

[0028] The molar concentration of the substrate is 10-200 mmol / L;

[0029] The organic solvent is a sulfur-containing organic solvent or an alcohol-based organic solvent;

[0030] The buffer solution is PBS, Tris-HCl, or Tris-H2SO4;

[0031] The reaction temperature is 0–50°C, and the reaction time is 16–30 hours;

[0032] The coenzyme regeneration system contains 1 mM NADP. + 20-400mM D-glucose, 0.1mg / mL-1mg / mL glucose dehydrogenase.

[0033] Furthermore, the organic solvent is dimethyl sulfoxide or methanol, and the buffer solution is PBS.

[0034] Furthermore, the reaction temperature is 17–47°C, and the reaction time is 16–24 hours.

[0035] Furthermore, the reaction temperature is 17°C, 27°C, or 37°C, and the reaction time is 16 hours, 18 hours, or 24 hours.

[0036] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0037] In the nucleotide mutation code of this invention, the numbers are the nucleotide site numbers of the mutation, the letters before the numbers represent the nucleotides before the mutation, and the letters after the numbers represent the nucleotides after the mutation. For example, GCA55-57GGA means that the nucleotides at positions 55, 56, and 57 are mutated from GCA to GGA, and so on.

[0038] In the amino acid mutation code of this invention, the numbers are the amino acid site numbers of the mutation, the letters before the numbers represent the amino acid before the mutation, and the letters after the numbers represent the amino acid after the mutation. For example, A19G means that the 19th amino acid is mutated from A to G, and so on.

[0039] In the amino acid mutation code of this invention, the letters before and after the numbers are amino acid abbreviations known in the art. For example, V represents Valine, A represents Alanine, M represents Methionine, L represents Leucine, Q represents Glutamine, C represents Cysteine, K represents Lysine, T represents Threonine, I represents Isoleucine, S represents Serine, H represents Histidine, E represents Glutamic acid, P represents Proline, F represents Phenylalanine, D represents Aspartic acid, Y represents Tyrosine, G represents Glycine, N represents Asparagine, W represents Tryptophan, R represents Arginine, M represents Methionine, and H represents Histidine.

[0040] In the combined mutation codes of this invention, "+" indicates a mutation performed before and after "+", and the symbols before and after " / " are synonymous codons for the amino acids resulting from the mutation at that site. For example, A19G+E278Y means that the 19th amino acid is mutated from A to G and the 278th amino acid is mutated from E to Y; GCA55-57GGA+GAG832-834TAC means that the 55th, 56th, and 57th nucleotides are mutated from GCA to GGA and the 832nd, 833rd, and 834th nucleotides are mutated from GAG to TAC; CAC736-738ATC / ATT means that the 736th, 737th, and 738th nucleotides are mutated from CAC to ATC or ATT, and so on.

[0041] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes. For example, the prefix Ca-b alkyl indicates any alkyl group containing "a" to "b" carbon atoms. For example, C1-6 alkyl refers to straight-chain or branched alkyl groups containing 1, 2, 3, 4, 5, or 6 carbon atoms.

[0042] In this invention, "substitution" refers to the replacement of one, two, or more hydrogen atoms in a molecule by other different atoms or molecules, including one, two, or more substitutions on isotopes or ectopic atoms in the molecule.

[0043] An aromatic ring refers to a fully carbon-cyclic group with a conjugated π-electron system, such as a phenyl group. The aryl group does not contain heteroatoms, and the point of attachment to the parent compound must be on a carbon atom of a ring with a conjugated π-electron system.

[0044] A heterocyclic aromatic ring refers to a ring containing one or more heteroatoms with a conjugated π-electron system.

[0045] Cyclic compounds composed of carbon atoms and heteroatoms are called heterocyclic compounds.

[0046] A saturated heterocyclic group refers to a heterocycle in which the bonds between the carbon atoms and heteroatoms on the ring are single bonds.

[0047] Heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen.

[0048] Halogens include F, Cl, Br, and I.

[0049] Integers from 1 to 10 include 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. And so on.

[0050] The present invention has achieved the following beneficial effects:

[0051] This invention uses the wild-type carbonyl reductase of *Stachybotrys chlorohalonata* as an evolutionary template and, through enzyme engineering, has obtained several mutants with significantly superior catalytic activity, thermal stability, and substrate spectra compared to the wild-type. The carbonyl reductase mutants of this invention can catalyze the production of various prochiral carbonyl compounds to chiral alcohols with high stereoselectivity (ee > 99%). These carbonyl reductase mutants provide a new, efficient, and environmentally friendly technical route for the industrial production of chiral alcohol products, possessing very high potential for industrial application.

[0052] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0053] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0054] Figure 1 This study assesses the temperature tolerance of the carbonyl reductase ScKRED mutant of this invention.

[0055] Figure 2 The chiral HPLC chromatograms of P1 and P2, the chiral products catalyzed by the carbonyl reductase ScKRED wild-type M0 and mutant M64 of this invention, are shown.

[0056] Figure 3 The chiral HPLC chromatograms of P4, the chiral product catalyzed by the carbonyl reductase ScKRED wild-type M0 and mutant M64 of this invention, are shown.

[0057] Figure 4 The chiral HPLC chromatograms of P3 and P5, the chiral products catalyzed by the carbonyl reductase ScKRED wild-type M0 and mutant M64 of this invention, are shown.

[0058] Figure 5 The chiral HPLC spectra of P7 and P8, the chiral products catalyzed by the carbonyl reductase ScKRED wild-type M0 and mutant M64 of this invention, are shown. Detailed Implementation

[0059] All raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products. The carbonyl reductase ScKRED involved in this invention was obtained from the NCBI database using genome mining technology. This gene is derived from Stachybotrys chlorohalonata (strain IBT 40285, uniprot: A0A084QW11). It has the DNA sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2.

[0060] The construction of the carbonyl reductase ScKRED expression vector in this invention involves the commonly used vectors pET-28a(+) and pGEX6p in the art. Specifically, the carbonyl reductase ScKRED gene is constructed on the pET-28a(+) vector with an N-terminal His tag for the preparation, purification, and activity assay of ScKRED lyophilized powder. For the construction and screening of mutant libraries, the ScKRED gene is constructed on the pGEX6p vector.

[0061] Heterologous expression of the carbonyl reductase ScKRED was performed in engineered E. coli strains. Specifically, a vector carrying the carbonyl reductase ScKRED gene was transformed into E. coli BL21(DE3) to construct recombinant bacteria. The extracted single clones were then cultured and induced to express the target protein, resulting in bacterial cells containing the target protein. TOP10 was used for plasmid construction, and BL21(DE3) was used for protein expression.

[0062] The preparation of carbonyl reductase lyophilized powder follows general methods in the art. Specifically, the recombinant bacteria induced and cultured are centrifuged for 10 min (6,000 rpm, 4℃), and the bacterial cells are collected. 1 g of bacterial cells is added to 7 mL of PBS phosphate buffer (pH 7.2-7.4, 0.01 M). The bacterial cells are disrupted by sonication, and the supernatant after centrifugation (12,000 rpm, 4℃, 20 min) is the crude enzyme solution. The crude enzyme solution is rapidly frozen with liquid nitrogen and then dried in a freeze dryer to obtain the lyophilized powder.

[0063] This invention relates to protein purification techniques. Specifically, the purification of the carbonyl reductase ScKRED, which contains an N-terminal histidine tag, is performed using affinity chromatography. The induced bacterial culture is resuspended in 10 mL Buffer A (1x PBS buffer), sonicated for 5 min, and the supernatant is collected by high-speed centrifugation. The supernatant is then purified by affinity chromatography, and the purified protein is processed using Buffer A. 280 The protein concentration was determined by nm to measure the protein solubility in an experiment.

[0064] The substrates involved in this invention are as follows:

[0065] Substrate S1 is Substrate S2 is Substrate S3 is Substrate S4 is Substrate S5 is Substrate S6 is Substrate S7 is Substrate S8 is Substrate S9 is Substrate S10 is Substrate S11 is Substrate S12 is Substrate S13 is

[0066] Example 1: Establishment and screening of a carbonyl reductase ScKRED mutant library

[0067] I. Construction and Screening of the SCKRED Random Mutant Library

[0068] This study utilized error-prone PCR to amplify the wild-type gene of ScKRED. The PCR product was chemically transformed into Top10 cells, and single colonies were picked and cultured in 96-well plates to construct a random mutant library. The specific screening method was as follows: the cell culture medium was centrifuged to remove the supernatant, lysozyme was added to each well, and the cells were incubated at 37°C for 1 h to lyse the cells, followed by incubation at 45°C for 20 min. After centrifugation, substrate S1, NADPH, and phosphate buffer (50 mM, pH 7.5) were added to the supernatant, and the solution was placed in a microplate reader. The absorbance of NADPH was measured at 30°C and 340 nm. A greater decrease in absorbance indicated higher residual activity of the mutant after incubation. By comparing the enzyme activity with that of the control group's carbonyl reductase, mutants with higher activity and stability were screened.

[0069] II. Selection of mutation sites in SKRED saturated libraries, construction and screening of saturated mutant libraries

[0070] Molecular docking software was used to simulate the molecular docking of the carbonyl reductase ScKRED protein structure with the substrate. Analysis revealed the following amino acid sites to interact with the substrate: L96, L98, Y174, S223, T224, P243, L238, S135, T136, S241, L242, E171, R172, Q245, H246, N209, P206, and S225. These sites were used as candidate sites to construct a saturated library. The saturated mutant library was constructed using the NDT / VHG / TGG codon combination, and the pGEX6p vector was used for construction. PCR products containing the mutants were obtained through conventional PCR amplification. These PCR products were then chemically transformed into *E. coli* top10 competent cells, and single clones containing the mutants were obtained on solid agar plates. Single clones were picked and cultured in 96-well plates for subsequent mutant library screening.

[0071] The construction and screening of saturated mutant libraries were divided into two rounds. In the first round, the ScKRED mutant with the mutation at position 19 being obtained by random screening (i.e., A19G, mutant number M1) was used as a template to construct and screen saturated libraries at positions L96, L98, Y174, S223, T224, P243, and L238.

[0072] The second round of screening used the ScKRED mutant (i.e., A19G+S207A+Q84K+A175I+E278F+A69Q+G122K+L96V+L238A, mutant number M22) carrying the mutations at positions 19-19 (G), 207-107 (A), 84-104 (K), 175-105 (I), 278-106 (F), 69-105 (Q), 122-105 (K), 96-106-106 (V), and 238-106 (A) as the template for constructing the second round of saturated libraries. S135, T136, S241, L242, E171, R172, Q245, H246, N209, P206, and S225 were constructed and screened.

[0073] The superior mutants obtained through the above screening are shown in Table 1.

[0074] Table 1. Preferred mutants of the carbonyl reductase SKRED obtained through screening.

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] In this invention, to ensure that the enzyme exhibits enhanced activity on different types of substrates during directed evolution and to maximize the enzyme's substrate spectrum, substrates S1 and S2, with different structures, were selected as screening substrates for library screening. Conversion rates were determined using HPLC to screen for mutants with high activity. Next, a library of beneficial mutant combinations was constructed using iterative strategies commonly used in the art to obtain even more advantageous mutants.

[0081] The following experimental examples demonstrate the beneficial effects of the present invention.

[0082] Experimental Example 1: Activity determination of the dominant mutant of carbonyl reductase ScKRED

[0083] The activities of the dominant mutants obtained in the screening of Example 1 on substrates S1 and S2 were determined, and the results are shown in Table 2.

[0084] The specific reaction setup for the activity assay in this experimental example is as follows:

[0085] Substrate S1 activity assay: The reaction system volume was 500 μL, containing 50 mM substrate S1, 7 mg / mL BcKRED mutant lyophilized powder, 1 mM NADP+, 100 mM D-glucose, 1 mg / mL NADPH regeneration system enzyme powder, 100 mM potassium phosphate buffer, pH 7.0 and 10% v / v DMSO.

[0086] Substrate S2 activity assay: The reaction system volume was 500 μL, containing 150 mM substrate S2, 1 mg / mL BcKRED mutant lyophilized powder, 1 mM NADP+, 300 mM D-glucose, 1 mg / mL NADPH regeneration system enzyme powder, 100 mM potassium phosphate buffer, pH 7.5 and 10% v / v DMSO;

[0087] The reaction solution was placed in a shaker (750 rpm, 30℃) and reacted for 18 h. After the reaction was completed, two volumes of acetonitrile were added to terminate the reaction. The supernatant was collected after centrifugation, and the conversion rate was detected by HPLC. The conversion rate was calculated based on the standard curve of the product. The catalytic activity was evaluated based on the conversion rate.

[0088] Table 2 Catalytic activities of the dominant carbonyl reductase SKRED mutant on substrates S1 and S2

[0089]

[0090]

[0091] Experiments have shown that most of the superior mutants of this invention have better catalytic activity than the wild-type M0.

[0092] Experimental Example 2: Determination of the melting temperature (Tm) of the dominant carbonyl reductase ScKRED mutant

[0093] The method used in this experimental example is a common method in the art. Specifically, the melting temperature (Tm) of the protein was determined using a CFX96 Real-Time PCR Detection System, with the program increasing the temperature from 25°C to 95°C in 0.5°C increments every 30 seconds. The specific reaction system used was as follows: 25 μL of the reaction system contained 0.4 mg / mL of purified 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.

[0094] Table 3. Stability determination of dominant mutants of carbonyl reductase SKRED

[0095]

[0096]

[0097] Experiments have shown that the superior mutants of this invention have higher Tm values ​​and better thermal stability compared to the wild type M0. Among them, the thermal melting temperature (Tm) of M63, M64, and M65 is increased to 63℃, which is 25.5℃ higher than that of the wild type. The mutants M67, M68, M69, and M70 are particularly outstanding, with Tm increased to 65℃-65.5℃, which is 28℃-28.5℃ higher than that of the wild type. They can adapt to higher temperature reaction conditions and have greater prospects for industrial application.

[0098] Experimental Example 3: Substrate spectrum of the dominant mutant of carbonyl reductase ScKRED

[0099] To evaluate whether the ScKRED mutants still possess broad substrate universality after improvements in activity and thermostability, this experiment measured the activities of the wild-type ScKRED and several mutants (M23, M28, M40, M48, M54, M59, M63, M64, M65, M66, M67) against substrates S3, S4, S5, S6, S7, S8, S9, S10, S11, and S12 with different structures. The activity measurement results are shown in Table 4.

[0100] Table 4. Activity results of ScKRED on different substrates

[0101]

[0102] Activity assay reaction setup: The reaction system volume was 500 μL, and 1 mM NADP was added. + The D-glucose concentration was twice the substrate equivalent, and the NADPH regeneration system consisted of 1 mg / mL enzyme powder, 100 mM potassium phosphate buffer (pH 7.5), and 10% v / v DMSO. Specific enzyme dosages and substrate concentrations are shown in Table 4. The conversion rates of substrates S1, S2, S3, S4, S5, S6, 7, S8, S9, and S12 were calculated using standard curves of the products. The conversion rates of substrates S10 and S11 were calculated using the peak area ratios of the product and substrate.

[0103] Experiments have shown that the multiple ScKRED mutants of this invention, while exhibiting improved thermal stability, possess higher catalytic activity for a variety of substrates than the wild-type ScKRED enzyme, demonstrating a broad substrate spectrum.

[0104] Experiment Example 4: Assessment of the temperature tolerance of carbonyl reductase ScKRED wild-type and mutant strains

[0105] This experiment selected mutants M28 (Tm = 57℃), M54 (Tm = 54℃), M64 (Tm = 63℃), and M67 (Tm = 65℃) with different degrees of increased Tm values ​​for temperature tolerance assessment. The specific implementation method is as follows.

[0106] Temperature incubation settings: 20 mg / mL of lyophilized ScKRED wild-type M0, M28, M54, M64, and M67 powders were dissolved in potassium phosphate buffer (pH 7.5, 0.1 M) and placed in water baths at 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C. Samples were taken after 0.5 h and 1 h for activity assay against substrate S13.

[0107] Reaction setup: A 500 μL reaction mixture was prepared in potassium phosphate buffer (pH 7.5, 0.1 M). Wild-type and mutant carbonyl reductases incubated at different temperatures were used. The reaction concentration was 5 mg / mL, with 50 mM substrate K3 and 1 mM NADP. + The reaction was carried out in an NADPH regeneration system with 10% DMSO. Reaction conditions: 30℃, 750 rpm, 16-18 h.

[0108] Incubation temperature activity test results are shown in the figure. Figure 1 .

[0109] Experiments have shown that the wild-type ScKRED (M0) has low temperature tolerance. After incubation at 45℃ for 1 hour, the conversion rate is only one-third of that at 30℃, and after incubation at 55℃ for 1 hour, the conversion rate is only 4% of that at 30℃, almost losing its catalytic activity. In contrast, the activities of the dominant ScKRED mutants M28, M54, M64, and M67 of this invention are almost unaffected by incubation at different temperatures compared to the conversion rate at 30℃, indicating that these mutants have significantly improved temperature tolerance. Other dominant mutants of this invention with higher Tm values ​​(Table 3) also exhibit similar temperature tolerance to the mutants in this experimental example.

[0110] Experimental Example 5: Determination of the selectivity of wild-type and mutant carbonyl reductase ScKRED for different substrates

[0111] This experimental example determined the selectivity preference of wild-type M0 and the thermally stable mutant M64 for the product alcohols generated from substrates S1, S2, S3, S4, S5, S7, and S8. The specific implementation method for chirality determination is described below.

[0112] Product chirality determination: The reaction system volume for chiral samples was 500 μL, containing 50 mM substrate and 1 mM NADP. + The reaction mixture consisted of D-glucose at a concentration of 2 equivalents of the substrate, 1 mg / mL of NADPH regeneration system enzyme powder, 10 mg / mL of MO or M64 lyophilized powder, 100 mM potassium phosphate buffer (pH 7.5), and 10% v / v DMSO. After the reaction was complete, a suitable amount of high-concentration NaOH solution was added, followed by 500 μL of MTBE (methyl tert-butyl ether) and 500 μL of water. After mixing and centrifugation, the MTBE layer was collected, extracted three times, and dried under vacuum to obtain the product alcohol. The configuration of the product was then determined by HPLC chiral column chromatography.

[0113] Table 5. Enantioselectivity results of ScKRED

[0114] S1 ＞99％(R) ＞99％(R) S2 ＞99％(R) ＞99％(R) S3 ＞99％(R) ＞99％(R) S4 ＞99％(R) ＞99％(R) S5 ＞99％(R) ＞99％(R) S7 90％(R) 95％(R) S8 ＞99％(R) 94％(R)

[0115] The results are shown in Table 5 and Figure 2-5 As shown, experiments have demonstrated that the carbonyl reductase ScKRED wild-type M0 and the mutant M64, which exhibits enhanced activity and stability, all show high R-selectivity (all > 90% R) for the tested substrates S1, S2, S3, S4, S5, S7, and S8.

[0116] In summary, this invention uses the wild-type carbonyl reductase of *Stachybotrys chlorohalonata* as an evolutionary template and, through enzyme engineering, has obtained several mutants with significantly superior catalytic activity, thermal stability, and substrate spectra compared to the wild-type. The carbonyl reductase mutants of this invention can catalyze the production of various prochiral carbonyl compounds to chiral alcohols with high stereoselectivity (ee > 99%). These carbonyl reductase mutants provide a new, efficient, and environmentally friendly technical route for the industrial production of chiral alcohol products, possessing very high potential for industrial application.

[0117] The nucleotide and amino acid sequences involved in this invention are as follows:

[0118] DNA sequence of SEQ ID NO.1 ScKRED

[0119]

[0120] The amino acid sequence of SEQ ID NO.2 SCKRED

[0121]

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is obtained by mutation based on the wild-type carbonyl reductase, the amino acid sequence of which 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, and 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, wherein the recombinant expression system is recombinant Escherichia coli.

6. 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.

7. Application of carbonyl reductase mutants in the catalytic production of chiral alcohols from substrates with asymmetric carbonyl structures, wherein the substrates with asymmetric carbonyl structures are... The carbonyl reductase mutant is obtained by mutation based on the wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO.

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

8. Application of carbonyl reductase mutants in the catalytic production of chiral alcohols from substrates with asymmetric carbonyl structures, wherein the asymmetric carbonyl structure substrate is: The carbonyl reductase mutant is obtained by mutation based on the wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO.

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

9. Application of carbonyl reductase mutants in the catalytic production of chiral alcohols from substrates with asymmetric carbonyl structures, wherein the asymmetric carbonyl structure substrate is: , , , , , , , , or The carbonyl reductase mutant is obtained by mutation based on the wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO.

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

10. Application of carbonyl reductase mutants in the catalytic production of chiral alcohols from substrates with asymmetric carbonyl structures, wherein the substrates with asymmetric carbonyl structures are: The carbonyl reductase mutant is obtained by mutation based on the wild-type carbonyl reductase, the amino acid sequence of which is shown in SEQ ID NO.

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

11. A method for preparing chiral alcohols, characterized in that, The method involves reacting a carbonyl reductase mutant, a substrate with an asymmetric carbonyl structure, an organic reagent, a reaction buffer, and a coenzyme regeneration system to obtain the product. The carbonyl reductase mutant is the carbonyl reductase mutant of claim 7, and the carbonyl asymmetric substrate is the carbonyl asymmetric substrate of claim 7; or, the carbonyl reductase mutant is the carbonyl reductase mutant of claim 8, and the carbonyl asymmetric substrate is the carbonyl asymmetric substrate of claim 8; or, the carbonyl reductase mutant is the carbonyl reductase mutant of claim 9, and the carbonyl asymmetric substrate is the carbonyl asymmetric substrate of claim 9; or, the carbonyl reductase mutant is the carbonyl reductase mutant of claim 10, and the carbonyl asymmetric substrate is the carbonyl asymmetric substrate of claim 10. The concentration of the carbonyl reductase mutant is 0.1-10 mg / mL; The molar concentration of the substrate is 10-200 mmol / L; The organic solvent is a sulfur-containing organic solvent or an alcohol-based organic solvent; The reaction buffer is PBS, Tris-HCl, or Tris-H2SO4; The reaction temperature is 30~55℃, and the reaction time is 16-30 hours; The coenzyme regeneration system contains 1 mM NADP. + 20-400 mM D-glucose, 0.1 mg / mL-1 mg / mL glucose dehydrogenase.

12. The method according to claim 11, characterized in that, The organic solvent is dimethyl sulfoxide or methanol; the reaction buffer is PBS.

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