Carbonyl reductase mutant as well as preparation method and application thereof

Abstract

The invention discloses a carbonyl reductase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, wild-type carbonyl reductase of Stachybotrys chlorohallonata is taken as an evolution template, and a plurality of mutants with catalytic activity, thermal stability and substrate spectrum obviously superior to those of the wild-type carbonyl reductase are obtained through an enzyme engineering technology. The carbonyl reductase mutant can catalyze various prochiral carbonyl compounds to obtain chiral alcohol with high stereoselectivity (ee is greater than 99%). The carbonyl reductase mutant provides a novel, efficient and environment-friendly technical route for industrial production of chiral alcohol products, and has very high industrial application potential.

Description

TECHNICAL FIELD

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

[0002] Ketoreductase (KRED), also known as carbonyl reductase (EC 1.1.1.184), is a kind of biological catalyst relying on NAD(H) / NADP(H) coenzyme for catalytic reaction, which can efficiently catalyze the asymmetric reduction reaction between aldehyde / ketone and chiral alcohol. The enzyme has become a key tool for the synthesis of chiral drug intermediates due to its excellent stereoselectivity, mild reaction conditions and environmental friendly characteristics. Carbonyl reductase is applied as a catalyst in the synthesis of corresponding intermediates in the synthesis of drugs such as ezetimibe, montelukast, duloxetine and atorvastatin. Compared with chemical methods, the enzyme catalytic synthesis method of chiral alcohol needs only one step from ketone compound to the corresponding alcohol compound, while the chemical method usually needs multiple steps to obtain the corresponding alcohol compound. Therefore, carbonyl reductase is increasingly widely used in industrial production.

[0003] Biocatalysis often needs to overcome relatively extreme environments in industrial production, such as high temperature, strong acid and alkali, organic solvent, etc. Enzyme as a catalyst usually needs to catalyze the reaction under relatively mild conditions, and the conditions in industrial production can easily lead to the inactivation of natural enzymes. Therefore, the development of KRED mutants with high thermal stability has become the research focus in the field of carbonyl reductase modification. In addition, in enzyme catalysis, the substrate spectrum of wild-type enzyme is usually narrow, and only specific types of substrates can be catalyzed to react, which limits its application in the industrial field.

[0004] Therefore, it is of important application value in industrial production to mine enzymes with broad substrate spectrum, improve their thermal stability, catalytic activity and substrate universality through enzyme engineering technology. SUMMARY

[0005] In order to solve the above problems existing in the prior art, the purpose of the present application is to provide a carbonyl reductase mutant and 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 a wild-type carbonyl reductase having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 2, wherein the mutation site is at least one of the following positions: 19th, 27th, 58th, 64th, 69th, 71st, 77th, 81st, 84th, 96th, 98th, 114th, 122nd, 124th, 135th, 136th, 158th, 175th, 207th, 216th, 238th, 241st, 242nd, 245th, 246th, 265th, 268th, 278th, 283rd, 289th, 295th, 331st, 336th and 337th.

[0008] Further, the wild-type carbonyl reductase has 90%, 95%, 96%, 97%, 98% or 99% or more homology with the amino acid sequence shown in SEQ ID NO. 2, and the mutation includes at least one of the following mutation modes: the amino acid residue at the 19th position is mutated to G, the amino acid residue at the 27th position is mutated to H, the amino acid residue at the 58th position is mutated to K, the amino acid residue at the 64th position is mutated to K, the amino acid residue at the 69th position is mutated to Q, the amino acid residue at the 71st position is mutated to P, the amino acid residue at the 77th position is mutated to G, the amino acid residue at the 81st position is mutated to E, the amino acid residue at the 84th position is mutated to K, the amino acid residue at the 96th position is mutated to V, the amino acid residue at the 98th position is mutated to F, the amino acid residue at the 114th position is mutated to L, the amino acid residue at the 122nd position is mutated to K, the amino acid residue at the 124th position is mutated to P, the amino acid residue at the 135th position is mutated to A, the amino acid residue at the 136th position is mutated to V, S or C, the amino acid residue at the 158th position is mutated to V, the amino acid residue at the 175th position is mutated to I, the amino acid residue at the 207th position is mutated to A, the amino acid residue at the 216th position is mutated to P or L, the amino acid residue at the 238th position is mutated to A, the amino acid residue at the 241st position is mutated to L, V or A, the amino acid residue at the 242nd position is mutated to G, the amino acid residue at the 245th position is mutated to N, C, L, I, V, K, T or M, the amino acid residue at the 246th position is mutated to W, D, R, I, M or T, the amino acid residue at the 265th position is mutated to D, the amino acid residue at the 268th position is mutated to N, the amino acid residue at the 278th position is mutated to F or Y, the amino acid residue at the 283rd position is mutated to I, the amino acid residue at the 289th position is mutated to K, the amino acid residue at the 295th position is mutated to T, the amino acid residue at the 331st position is mutated to E, the amino acid residue at the 336th position is mutated to R, and the amino acid residue at the 337th position is mutated to A.

[0009] Further, the amino acid sequence of the wild-type carbonyl reductase is shown as SEQ ID NO. 2, and the mutation comprises at least one of the following unit point mutations or combined mutations:

[0010]

[0011]

[0012]

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

[0014] The application also provides a recombinant expression vector comprising the above-mentioned gene, which is selected from a plasmid, an artificial chromosome, a bacteriophage or a viral vector.

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

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

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

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

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

[0020] (2) transforming the vector into a host bacterium, culturing, adding an inducer, and inducing the expression of the carbonyl reductase mutant.

[0021] The application also provides the use of the above-mentioned carbonyl reductase mutant in catalyzing the asymmetric substrate with a carbonyl structure to produce chiral alcohol. m and n are each independently selected from 0 or 1; R1 and R2 are not the same, and R1 and R2 are each independently selected from C1-9 alkyl, C1-9 alkoxy, heterocyclic group, cycloalkyl, aromatic ring, heteroaromatic ring; or, R1 and R2 are each independently selected from substituted C1-9 alkyl, C1-9 alkoxy, heterocyclic group, cycloalkyl, aromatic ring, heteroaromatic ring, and each of the substituents is 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 connected to form a ring;

[0022] Further, the asymmetric substrate with a carbonyl structure is wherein R3, R4, R5, R6, R7 are each independently selected from H, halogen, C1-6alkyl, C1-6alkoxy, halogen-substituted C1-6alkyl, halogen-substituted C1-6alkoxy, R8 is H, halogen, C1-6alkyl, halogen-substituted C1-6alkyl or R9 is C1-6alkyl or halogen-substituted phenyl, R 10 is halogen, R 11 is C1-6alkyl or benzyl, a, b, c are each independently selected from an integer from 1 to 10.

[0023] Further, R3 is H or Cl, R4 is H, F, CI, Br or CH3, R5 is H, F, CI, CF3 or methoxy, R6 is H, CI or F, R7 is H, F, CI, CH3 or CH2CI, R8 is H, CF3, CH2CI, Br or R9 is CH3 or C6H4CI, R 10 is Br, R 11 is C1-6alkyl or benzyl, a, b, c are each independently selected from an integer from 1 to 5.

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

[0025]

[0026] The application also provides a method for preparing chiral alcohol, which comprises reacting the above-mentioned carbonyl reductase mutant, substrate with asymmetric carbonyl structure, organic reagent, reaction buffer solution and coenzyme regeneration system.

[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 sulfur-containing organic solvent or alcohol organic solvent.

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

[0031] The temperature of the reaction is 0-50℃, and the reaction time is 16-30 hours.

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

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

[0034] Further, the temperature of the reaction is 17-47℃, and the time of the reaction is 16-24 hours.

[0035] Further, the temperature of the reaction is 17℃, 27℃ or 37℃, and the time of the reaction is 16 hours, 18 hours or 24 hours.

[0036] Definitions of terms used in the present application: Unless otherwise indicated, the initial definition of a group or term provided herein applies throughout the specification; for terms not specifically defined herein, the meaning given to them by one of ordinary skill in the art in light of the disclosure and context will prevail.

[0037] 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, GCA55-57GGA means that the 55th, 56th and 57th nucleotides are mutated from GCA to GGA, and so on.

[0038] 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, A19G means that the 19th amino acid is mutated from A to G, and so on.

[0039] In the amino acid mutation code of the present application, the letters before and after the number are the amino acid abbreviations 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), N represents Asparagine (Asparagine), W represents Tryptophan (Tryptophan), R represents Arginine (Arginine), M represents Methionine (Methionine), H represents Histidine (Histidine).

[0040] In the combination mutation code of the present application, "+" indicates that mutations before "+" and after "+" are performed, and " / " indicates synonymous codons of the amino acids after mutation of the site. For example, A19G+E278Y indicates 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 indicates 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 indicates that the 736th, 737th, and 738th nucleotides are mutated from CAC to ATC or ATT, and the like.

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

[0042] The "substitution" of the present application refers to the replacement of 1, 2, or multiple hydrogen atoms in a molecule with other different atoms or molecules, including 1, 2, or multiple substitutions of isotopic atoms or heteroatoms in the molecule.

[0043] Aromatic ring refers to a fully carbon ring group having a conjugated pi electron system, for example, phenyl. The aromatic group does not contain heteroatoms, and the point of attachment to the parent must be on a carbon atom on the ring having a conjugated pi electron system.

[0044] Heteroaromatic ring refers to a ring having a conjugated pi electron system containing one to multiple heteroatoms.

[0045] A cyclic compound composed of carbon atoms and heteroatoms is referred to as a heterocyclic compound.

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

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

[0048] Halogen includes F, Cl, Br, and I.

[0049] The integer of 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The same applies to the following.

[0050] The present application has the following beneficial effects:

[0051] The present application takes wild-type carbonyl reductase of Stachybotrys chlorohalonata as an evolutionary template, and obtains a plurality of mutants with catalytic activity, thermal stability and substrate spectrum superior to the wild type through enzyme engineering technology. The carbonyl reductase mutant of the present application can catalyze a variety of prochiral carbonyl compounds to obtain chiral alcohol with high stereoselectivity (ee>99%). The carbonyl reductase mutant of the present application provides a new, efficient and environmentally friendly technical route for the industrial production of chiral alcohol products, and has very high industrial application potential.

[0052] 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 modification, replacement or change can be made without departing from the above basic technical idea of the present application.

[0053] The above content of the present application will be further described in detail through the specific embodiments. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following embodiments. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The temperature tolerance of the carbonyl reductase ScKRED mutant of the present application was evaluated.

[0055] Figure 2 The chiral HPLC analysis spectrum of the chiral products P1 and P2 catalyzed by the wild type M0 and the mutant M64 of the carbonyl reductase ScKRED of the present application.

[0056] Figure 3 The chiral HPLC analysis spectrum of the chiral product P4 catalyzed by the wild type M0 and the mutant M64 of the carbonyl reductase ScKRED of the present application.

[0057] Figure 4 The chiral HPLC analysis spectrum of the chiral products P3 and P5 catalyzed by the wild type M0 and the mutant M64 of the carbonyl reductase ScKRED of the present application.

[0058] Figure 5 The chiral HPLC analysis spectrum of the chiral products P7 and P8 catalyzed by the wild type M0 and the mutant M64 of the carbonyl reductase ScKRED of the present application. DETAILED DESCRIPTION

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

[0060] The construction of the carbonyl reductase ScKRED expression vector in the present application involves the commonly used vectors pET-28a(+) and pGEX6p in the art. Specifically, the carbonyl reductase ScKRED gene is constructed in the pET-28a(+) vector with an N-terminal His tag, which is used for the preparation, purification and activity determination of the ScKRED lyophilized powder. In order to construct and screen the mutant library, the ScKRED gene is constructed in the pGEX6p vector.

[0061] The heterologous expression of the carbonyl reductase ScKRED is carried out in the engineered E. coli. Specifically, the vector carrying the carbonyl reductase ScKRED gene is transformed into E. coli BL21(DE3) to construct a recombinant bacterium. Then the extracted monoclonal is cultured and induced for expression to obtain a bacterium containing the target protein. TOP10 is selected in the plasmid construction stage, and BL21(DE3) is selected in the protein expression stage.

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

[0063] The present application involves protein purification technology. Specifically, the carbonyl reductase ScKRED containing an N-terminal histidine tag is purified by affinity chromatography. The induced cultured bacteria are resuspended in 10 mL Buffer A (1x PBS buffer), ultrasonically broken for 5 min, and the supernatant is collected by high-speed centrifugation. Then the supernatant is purified by affinity chromatography, and the purified protein is determined by A 280 nm to determine the protein solubility.

[0064] The substrate involved in the present application is as follows:

[0065] The substrate S1 is The 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 mutant library of carbonyl reductase ScKRED

[0067] I. Construction and screening of ScKRED random mutation library

[0068] In this study, the wild type gene of ScKRED was amplified by error-prone PCR, and the PCR product was transformed into Top10 cells using a chemical method. Single colony bacteria were selected and cultured in 96-well plates to construct a random mutation library. The specific screening method is as follows: centrifuge the cell culture solution to remove the supernatant, add lysozyme to each well, incubate at 37°C for 1 h to break, then incubate at 45°C for 20 min, centrifuge, and then add substrate S1, NADPH and phosphate buffer (50 mM, pH 7.5) to the supernatant. Place in a microplate reader and detect the absorbance change of NADPH at 30°C and 340 nm wavelength. The greater the decrease in absorbance, the higher the residual activity of the mutant after temperature incubation. By comparing the enzyme activity with the control group carbonyl reductase, the mutant strain with higher activity and stability was selected.

[0069] II. Selection of ScKRED saturated library mutation sites, construction and screening of saturated mutant library

[0070] The protein structure of the carbonyl reductase ScKRED is subjected to molecular docking simulation with the substrate by using molecular docking software. Through analysis, it is presumed that the amino acid sites interacting with the substrate are: L96, L98, Y174, S223, T224, P243, L238, S135, T136, S241, L242, E171, R172, Q245, H246, N209, P206, S225. These sites are used as candidate sites to construct a saturation library. The saturation mutant library is constructed by using the codon combination of NDT / VHG / TGG, and the construction vector of the saturation library is pGEX6p vector. The PCR product containing the mutation is obtained by conventional PCR amplification, and then the PCR product is transferred into E. coli top10 competent cells by chemical method, and single colonies containing mutants are obtained in solid plate medium. The single colonies are picked and cultured in 96-well plates for subsequent mutant library screening.

[0071] The construction and screening of the saturation mutant library are divided into two rounds. In the first round, the ScKRED mutant containing the 19th mutation G (i.e. A19G, mutant number M1) obtained by random screening is used as a template to construct and screen the saturation library of L96, L98, Y174, S223, T224, P243, L238 sites.

[0072] In the second round of screening, the ScKRED mutant carrying the 19th mutation G, the 207th mutation A, the 84th mutation K, the 175th mutation I, the 278th mutation F, the 69th mutation Q, the 122nd mutation K, the 96th mutation V, and the 238th mutation A (i.e. A19G+S207A+Q84K+A175I+E278F+A69Q+G122K+L96V+L238A, mutant number M22) is used as a template for the construction of the second round of saturation library to construct and screen the saturation library of S135, T136, S241, L242, E171, R172, Q245, H246, N209, P206, S225 sites.

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

[0074] Table 1: Superior mutants of carbonyl reductase ScKRED obtained by screening

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] In the present application, in order to ensure that the enzyme has activity for different types of substrates in the process of directed evolution, and to maximize the expansion of the substrate spectrum of the enzyme, different substrates S1 and S2 are selected as screening substrates for library screening. The conversion rate is determined by HPLC, and mutants with high activity are screened. Next, a combination library of beneficial mutations is constructed using the iterative strategy commonly used in the art to obtain more advantageous mutants.

[0081] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0082] Experimental Example 1: Activity determination of carbonyl reductase ScKRED advantage mutants

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

[0084] The specific reaction settings for activity determination in this experimental example are as follows:

[0085] Substrate S1 activity determination: the reaction system volume was 500 μL, containing 50 mM substrate S1, 7 mg / mL BcKRED mutant freeze-dried 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 determination: the reaction system volume was 500 μL, containing 150 mM substrate S2, 1 mg / mL BcKRED mutant freeze-dried 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°C) and reacted for 18 h. After the reaction was completed, two volumes of acetonitrile were added to terminate the reaction, and after centrifugation, the supernatant was taken and the conversion rate was detected by HPLC. The conversion rate was calculated according to the standard curve of the product. The catalytic activity was evaluated according to the conversion rate.

[0088] Table 2 Catalytic activity of carbonyl reductase ScKRED advantage mutants on substrates S1 and S2

[0089]

[0090]

[0091] Experiments prove that most of the advantageous mutants of the application have better catalytic activity than the wild type M0.

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

[0093] The method used in this experimental example is a general method in the art. Specifically, the melting temperature (Tm) of the protein is determined by CFX96 Real-Time PCR Detection System, and the program is to raise the temperature from 25℃ to 95℃, and the temperature is raised by 0.5℃ every 30s. The specific reaction system of the sample used is: 25μL reaction system contains 0.4mg / mL purified protein, 10x fluorescence dye (5000X, SYPRO Orange dye), and the buffer is 1x PBS. The Tm value determination results of the mutants are shown in Table 3. TM Orange dye), and the buffer is 1x PBS. The Tm value determination results of the mutants are shown in Table 3.

[0094] Table 3: Stability determination of the carbonyl reductase ScKRED advantageous mutants

[0095]

[0096]

[0097] Experiments prove that the advantageous mutants of the application have higher Tm values and better thermal stability than the wild type M0. Among them, the thermal melting temperature Tm of M63, M64 and M65 is increased to 63℃, which is increased by 25.5℃ compared with the wild type; the particularly outstanding mutants M67, M68, M69 and M70 have a Tm of 65℃-65.5℃, which is increased by 28℃-28.5℃ compared with the wild type, and can adapt to relatively high temperature reaction conditions, and have more industrial application prospects.

[0098] Experimental Example 3: Substrate spectrum of the carbonyl reductase ScKRED advantageous mutants

[0099] In order to evaluate whether the ScKRED mutants still have wide substrate universality after the activity and thermal stability are improved, the activity of the wild type and multiple mutants M23, M28, M40, M48, M54, M59, M63, M64, M65, M66, M67 of ScKRED on substrates S3, S4, S5, S6, S7, S8, S9, S10, S11 and S12 with different structures is determined. The activity determination results are shown in Table 4.

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

[0101]

[0102] Activity assay reaction setup: 500 μL reaction volume, 1 mM NADP + , 2 equivalents of substrate D-glucose, 1 mg / mL NADPH regenerating system enzymes, 100 mM potassium phosphate buffer, pH 7.5 and 10% v / v DMSO; enzyme amount and substrate concentration are listed in Table 4. Conversion of substrates S1, S2, S3, S4, S5, S6, S7, S8, S9 and S12 were calculated using standard curves of the products. Conversion of substrates S10 and S11 were calculated using the ratio of peak area of product to substrate.

[0103] Experiments proved that the multiple ScKRED mutants of the application have higher catalytic activity than the ScKRED wild-type enzyme on a variety of substrates on the basis of improved thermal stability, and have a wide substrate spectrum.

[0104] Experimental Example 4: Temperature tolerance evaluation of carbonyl reductase ScKRED wild-type and mutants

[0105] This experimental example selected mutants M28 (Tm = 57℃), M54 (Tm = 54℃), M64 (Tm = 63℃) and M67 (Tm = 65℃) with different degrees of improved Tm values for temperature tolerance evaluation, and the specific implementation is described as follows.

[0106] Temperature incubation setup: 20 mg / mL ScKRED wild-type M0, M28, M54, M64 and M67 lyophilized powder was dissolved in potassium phosphate buffer (pH 7.5, 0.1 M) and placed in a water bath at 30℃, 35℃, 40℃, 45℃, 50℃ and 55℃. After incubation for 0.5 h and 1 h, samples were taken for activity assay on substrate S13.

[0107] Reaction setup: 500 μL reaction volume was set up in potassium phosphate buffer (pH 7.5, 0.1 M), and different temperature incubated carbonyl reductase wild-type and mutants were taken for reaction at a concentration of 5 mg / mL, 50 mM substrate K3, 1 mM NADP + , NADPH regenerating system, 10% DMSO. Reaction conditions: 30℃, 750 rpm, 16-18 h.

[0108] Incubation temperature activity assay results are shown in Figure 1 .

[0109] The experiment proves that the wild type (M0) of ScKRED has low tolerance to temperature, and after incubation at 45°C for 1 h, the conversion rate is only one third of that at 30°C, and after incubation at 55°C for 1 h, the conversion rate is only 4% of that at 30°C, and almost loses catalytic activity. The activities of the advantageous mutants M28, M54, M64 and M67 of the carbonyl reductase ScKRED of the application are almost not affected after incubation at different temperatures compared with the conversion rate at 30°C, indicating that the temperature tolerance of these mutants has been significantly improved. The other advantageous mutants with high Tm values (Table 3) of the application can also exhibit similar temperature tolerance to the mutants in this experimental example.

[0110] Experimental Example 5: Determination of the selectivity of wild type and mutants of carbonyl reductase ScKRED to different substrates

[0111] This experimental example determines the selectivity preference of the wild type M0 and the mutant M64 with high thermal stability to the product alcohol generated by catalyzing substrates S1, S2, S3, S4, S5, S7, S8. The chiral determination is specifically implemented as follows.

[0112] Chiral sample reaction system: 500 μL, 50 mM substrate, 1 mM NADP + , 2 equivalents of D-glucose, 1 mg / mL NADPH regenerating system enzyme powder, 10 mg / mL M0 or M64 freeze-dried powder, 100 mM potassium phosphate buffer, pH 7.5 and 10% v / v DMSO. After the reaction is completed, an appropriate amount of high-concentration NaOH solution is added, then 500 μL MTBE (methyl tert-butyl ether) and 500 μL water are added, mixed and centrifuged, the MTBE layer is taken, extracted three times, vacuum dried to obtain the product alcohol, and then the configuration of the product is detected by HPLC chiral column.

[0113] Table 5. Enantiomeric selectivity results of ScKRED

[0114] Substrate M0 M64 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] As shown in Table 5 and Figures 2-5 , the experiment proves that the wild type M0 of carbonyl reductase ScKRED and the mutant M64 with improved activity and stability both exhibit high R-selectivity (all > 90% R) to the determined substrates S1, S2, S3, S4, S5, S7, S8.

[0116] In summary, the present application takes wild-type carbonyl reductase of Stachybotrys chlorohalonata as an evolutionary template, and obtains multiple mutants with catalytic activity, thermal stability and substrate spectrum superior to the wild type through enzyme engineering technology. The carbonyl reductase mutants of the present application can catalyze a variety of prochiral carbonyl compounds to obtain chiral alcohol with high stereoselectivity (ee>99%). The carbonyl reductase mutants of the present application provide a new, efficient and environmentally friendly technical route for the industrial production of chiral alcohol products, and have very high industrial application potential.

[0117] The nucleotide sequence and amino acid sequence involved in the present application are as follows:

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

[0119]

[0120] SEQ ID NO. 2 Amino acid sequence of 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, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO.

2. The mutation 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.

2. The carbonyl reductase mutant according to claim 1, wherein The wild-type carbonyl reductase has 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence shown in SEQ ID NO.

2. The mutation includes at least one of the following mutation methods: mutating the 19th amino acid residue to G, the 27th amino acid residue to H, the 58th amino acid residue to K, the 64th amino acid residue to K, the 69th amino acid residue to Q, the 71st amino acid residue to P, the 77th amino acid residue to G, the 81st amino acid residue to E, the 84th amino acid residue to K, the 96th amino acid residue to V, the 98th amino acid residue to F, the 114th amino acid residue to L, the 122nd amino acid residue to K, the 124th amino acid residue to P, the 135th amino acid residue to A, and the 136th amino acid residue to V, S, or C. 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.

3. The carbonyl reductase mutant of claim 2, wherein 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 combinations of mutations:

4. A gene encoding the carbonyl reductase mutant of any one of claims 1-3.

5. A recombinant expression vector comprising the gene of claim 4, wherein the recombinant expression vector is selected from the group consisting of a plasmid, an artificial chromosome, a bacteriophage or a viral vector; preferably, the plasmid is a pET-21a vector or a pGEx6p vector.

6. A recombinant expression system comprising the recombinant expression vector of claim 5; preferably, the recombinant expression system is a recombinant Escherichia coli.

7. A method for preparing the carbonyl reductase mutant according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) constructing a vector containing a gene encoding the carbonyl reductase mutant of any one of claims 1-3; (2) transforming the vector into a host bacterium, culturing, adding an inducer, and inducing the expression of the carbonyl reductase mutant.

8. Use of a carbonyl reductase mutant according to any one of claims 1 to 3 for catalyzing the production of chiral alcohols from a substrate with an asymmetric carbonyl structure, said substrate with an asymmetric carbonyl structure being ###0003### m, n are each independently selected from 0 or 1 ; R1 is not identical to R2 and R1 and R2 are each independently selected from C1-9 alkyl, C1-9 alkoxy, heterocyclyl, cycloalkyl, aryl ring, heteroaryl ring; or, R1 and R2 are each independently selected from substituted C1-9 alkyl, C1-9 alkoxy, heterocyclyl, cycloalkyl, aryl ring, heteroaryl ring, said 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, R2 are linked to form a ring; Preferably, the substrate having an asymmetric carbonyl structure is wherein, R3, R4, R5, R6, R7are each independently selected from H, halogen, Ci-6alkyl, Ci-6alkoxy, halogen substituted Ci-6alkyl, halogen substituted Ci-6alkoxy, R8is H, halogen, Ci-6alkyl, halogen substituted Ci-6alkyl or R9is Ci-6alkyl or halogen substituted phenyl, R 10 is halogen, R 11 is Ci-6alkyl or benzyl, a, b, c are each independently selected from an integer between 1 and 10; preferably, R3is H or CI, R4is H, F, CI, Br or CH3, R5is H, F, CI, CF3or methoxy, R6is H, CI or F, R7is H, F, CI, CH3or CH2CI, R8is H, CF3, CH2CI, Br or R9is CH3or C6H4CI, R 10 is Br, R 11 is Ci-6alkyl or benzyl, a, b, c are each independently selected from an integer between 1 and 5.

9. Use according to claim 8, characterized in that, The asymmetric substrate with a carbonyl structure is:

10. A process for the preparation of a chiral alcohol, characterized in that, The method is to react the carbonyl reductase mutant of claims 1-3, the asymmetric substrate with a carbonyl structure of claims 8 or 9, an organic reagent, a reaction buffer and a coenzyme regeneration system, and then obtain the product. 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 organic solvent, preferably dimethyl sulfoxide or methanol. The buffer solution is PBS, Tris-HCl or Tris-H2SO4, preferably PBS. The temperature of the reaction is 0-50°C, and the reaction time is 16-30 hours. The coenzyme regeneration system comprises 1 mM NADP + 20-400 mM D-glucose, 0.1 mg / mL - 1 mg / mL glucose dehydrogenase.

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