Carbonyl reductase mutant for synthesizing chiral alcohol sulfone compound and application of carbonyl reductase mutant

Abstract

The invention discloses a carbonyl reductase mutant for synthesizing a chiral alcohol sulfone compound and application of the carbonyl reductase mutant, a mutant M4A1 of carbonyl reductase ChKRED20 is used as a female parent, serine at the 188th site is mutated into any one of asparagine, glycine or threonine, and the carbonyl reductase mutant for synthesizing the chiral alcohol sulfone compound is obtained. Specifically, a mutant M4A1 of ChKRED20 is taken as a female parent, serine at the 188th site is mutated into asparagine, and a mutant ChKRED20-M4C4 is obtained. The mutant M4A1 of ChKRED20 is used as a female parent, serine at the 188th site is mutated into glycine, and the mutant ChKRED20-M4C3 is obtained. The mutant M4A1 of ChKRED20 is used as a female parent, serine at the 188th site is mutated into threonine, and the mutant ChKRED20-M4C6 is obtained. The three mutants can be used as catalysts to synthesize chiral alcohol sulfone compounds, and especially have great advantages in asymmetric synthesis of alpha-substituted beta-alcohol sulfone.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and in particular to a carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds and its application. Background Art

[0002] Chiral alcohol sulfone compounds are important structural units in organic chemistry and are widely used in the synthesis of drug molecules and bioactive compounds such as γ-butenolide, γ-butyrolactone, etc. In recent years, researchers have developed various methods for synthesizing chiral alcohol sulfone compounds, including asymmetric conjugate boration of α,β-unsaturated sulfones, asymmetric reduction strategies of α-substituted β-ketosulfones such as borane reduction, transition metal-catalyzed asymmetric hydrogenation, and asymmetric transfer hydrogenation. However, these methods generally have problems such as harsh high-pressure operating conditions, dependence on complex chiral ligand catalysts, or heavy metal pollution. In addition, α-substituted β-hydroxy sulfones contain two chiral centers, and it is difficult for traditional catalysts to precisely control the configuration of multiple chiral centers. Therefore, it is of great significance to develop an environmentally friendly and sustainable biocatalytic pathway for synthesizing such compounds. Enzyme catalysis has received extensive attention from researchers due to its excellent selectivity and specificity.

[0003] In recent years, the dynamic reduction dynamic kinetic resolution (DYRKR) technology catalyzed by ketoreductase (KRED) has become an important means for synthesizing compounds with multiple chiral centers. The increasing wide application of this biocatalytic strategy not only stems from its inherent advantages such as operational sustainability and mild reaction conditions, but more fundamentally lies in its ability to generate only a single stereoisomer from four possible configurations in a single enzyme-catalyzed step, while achieving complete conversion of the substrate. In the substrate spectrum of KRED-catalyzed DYRKR, enzymatic asymmetric synthesis of α-substituted β-alcohol esters, thioesters, nitriles, phosphonates, and amides has been reported. However, to date, the research on KRED-catalyzed DYRKR of α-substituted β-ketosulfone compounds is still extremely scarce, and there is no relevant research report on the efficient synthesis of chiral α-substituted β-hydroxy sulfones through KRED. There are reports in the prior art on the synthesis of chiral alcohols using carbonyl reductase, but these carbonyl reductases cannot achieve the synthesis of chiral alcohol sulfone compounds. There are probably two main reasons: one is that the existing carbonyl reductases cannot precisely regulate such compounds with multiple chiral centers; the other is that the volume of such substrates is large, and the existing enzymes cannot achieve the reduction of such substrates. Summary of the Invention

[0004] Aiming at the technical problem that the existing carbonyl reductase cannot synthesize chiral alcohol sulfone compounds, the present invention provides a carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds and its application.

[0005] Based on the previous research on the carbonyl reductase ChKRED20, more in-depth protein modification was carried out on it to obtain three carbonyl reductase mutants. Specifically, using the mutant M4A1 of ChKRED20 (abbreviated as ChKRED20-M4A1) as the parent, a single-point mutation was made on serine at position 188 to obtain the following three mutants:

[0006] ChKRED20-M4C3 (Q97L+S153L+Y188G+H145A),

[0007] ChKRED20-M4C4 (Q97L+S153L+Y188N+H145A),

[0008] ChKRED20-M4C6 (Q97L+S153L+Y188T+H145A).

[0009] Using these three mutants as catalysts can achieve the asymmetric synthesis of 16 β-hydroxy sulfones and α-substituted β-hydroxy sulfones.

[0010] The parent ChKRED20-M4A1 was obtained by using the amino acid sequence of the carbonyl reductase mutant strain ChKRED20-M2 (Q97L / S153L) as the starting sequence, mutating tyrosine at position 188 to serine, and mutating histidine at position 145 to alanine. The specific construction method can be found in the description of Patent CN202410908168.6.

[0011] The construction method of the mutant ChKRED20-M4C4 is as follows:

[0012] Using ChKRED20-M4A1 as the parent, serine at position 188 was mutated to asparagine to obtain the mutant ChKRED20-M4C4. Embodiment: Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer: 5’-CGGTGGGGCCTGCTAATATTGAAACCC-3’ (Sequence Listing SEQ ID No: 1) and the reverse primer: 5’-GGGTTTCAATATTAGCAGGCCCCACCG-3’ (Sequence Listing SEQ ID No: 2) were selected. The single-point mutation of serine at position 188 was carried out by using PCR technology to obtain the crude enzyme solution of ChKRED20-M4C4.

[0013] The construction method of the mutant ChKRED20-M4C3 is as follows:

[0014] Using ChKRED20-M4A1 as the female parent, serine at position 188 was mutated to glycine to obtain the mutant ChKRED20-M4C3. Implementation scheme: Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer was selected: 5’-T G G GG C C T G C T G G C A T T G A A A C C C C G C T G T TGGAA-3’ (Sequence Listing SEQ ID No: 3), and the reverse primer: 5’-TTCCAACAGCGGGGTTTCAATGCCA GCAGGCCCCA-3’ (Sequence Listing SEQ ID No: 4). The single-point mutation of serine at position 188 was carried out using PCR technology to obtain the crude enzyme solution of ChKRED20-M4C3.

[0015] The construction method of the mutant ChKRED20-M4C6 is as follows:

[0016] Using ChKRED20-M4A1 as the female parent, serine at position 188 was mutated to threonine to obtain the mutant ChKRED20-M4C6. Implementation scheme: Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer was selected: 5’-AATGCGGTGGGGCCTGCTACCATTGAAACCCC-3’ (Sequence Listing SEQ ID No: 5), and the reverse primer: 5’-GGGGTTTCAATGGTAGCAGGCCCCACCGCATT-3’ (Sequence Listing SEQ ID No: 6). The single-point mutation of serine at position 188 was carried out using PCR technology, and the crude enzyme solution of ChKRED20-M4C6 was obtained through protein expression.

[0017] The present invention also provides the applications of the above mutants ChKRED20-M4C4, ChKRED20-M4C3, and ChKRED20-M4C6. Any one of the three mutants can be used as a catalyst to catalyze the synthesis of chiral alcohol sulfone compounds. The synthesis method is as follows:

[0018] The substrate compound and the carbonyl reductase mutant (catalyst) for synthesizing chiral alcohol sulfone compounds were dispersed in a PBS buffer solution containing β-nicotinamide adenine dinucleotide (NAD+), and isopropanol was added, and then the reaction was carried out at 35 - 40 °C for 10 - 16 h to obtain the chiral alcohol sulfone compounds.

[0019] Among them, the mutant ChKRED20-M4C4 is used to catalyze the synthesis of any one of the following chiral alcohol sulfone compounds:

[0020]

[0021] In the formula, R1 is one of -H, -F, -Cl, -Me; R2 is one of -H, -F, -Cl, -Me; R3 is -CH3 or -Cl; R4 is one of -CH2CH3, cyclopropyl, -N(CH3)2.

[0022] The mutant ChKRED20-M4C6 is used for catalytic synthesis of the following compound:

[0023]

[0024] The mutant ChKRED20-M4C3 is used for catalytic synthesis of the following compound:

[0025]

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) Through the optimization of the synthesis route, the present invention obtains three mutants, ChKRED20-M4C3, ChKRED20-M4C4, and ChKRED20-M4C6, by single-point mutation, and these three mutants can achieve the efficient synthesis of chiral alcohol sulfones. For example, the asymmetric synthesis of 16 kinds of β-hydroxy sulfones and α-substituted β-hydroxy sulfones is realized as recorded in the examples of the present invention. There is no report in the prior art on the asymmetric synthesis of chiral alcohol sulfone compounds using carbonyl reductase or alcohol dehydrogenase. At the same time, the present invention reports for the first time the dynamic kinetic resolution of α-substituted β-ketosulfones.

[0028] (2) The mutant strain of carbonyl reductase ChKRED20 used in the present invention has a very broad substrate spectrum (ACSCatalysis 2024, 17480-17488; Org.Chem.Front. 2024, 11, 1804-1810; Synfacts 2025, 181-181.). Compared with carbonyl reductases or short-chain dehydrogenases of the same category with excellent catalytic performance, the catalytic efficiency and selectivity of the ChKRED20 mutant are higher, and different ChKRED20 mutant strains can be applied to a variety of different types of compounds.

[0029] (3) Most of the reported carbonyl reductase or short-chain dehydrogenase catalytic systems in the prior art need to use a coenzyme recycling system (glucose dehydrogenase / glucose) to regenerate cofactors NADH / NADPH, while the ChKRED20 mutant catalytic system of the present application does not require a coenzyme recycling system. Only a small amount of isopropanol can efficiently regenerate NADH, greatly simplifying the biocatalytic reaction system (an aqueous reaction system containing 10% volume fraction of isopropanol), which is beneficial to the subsequent treatment of the reaction.

[0030] Other advantages, objects and features of the present invention will be partly embodied by the following description, and partly will be understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0031] Figure 1 It is the 1H NMR spectrum of racemate 1b.

[0032] Figure 2 It is the liquid chromatography spectrum of the catalytic synthesis of (1S,2S)-1b by ChKRED20-M4C4.

[0033] Figure 3 It is the synthesis result data of various chiral alcohol sulfone compounds.

[0034] Figure 4 It is the 1H NMR spectrum of racemate 11b.

[0035] Figure 5 It is the liquid chromatography spectrum of the catalytic synthesis of (1S,2S)-11b by ChKRED20-M4C6.

[0036] Figure 6 It is the product morphology diagram of (1S,2R)-12b obtained from the scale-up reaction.

[0037] Figure 7 It is the 1H NMR spectrum of racemate 12b.

[0038] Figure 8 It is the liquid chromatography spectrum of the catalytic synthesis of (1S,2R)-12b by ChKRED20-M4C3. Detailed Embodiments

[0039] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] Example 1

[0041] Construction of mutant ChKRED20-M4C4:

[0042] Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer: 5’-CGGTGGGGCCTGCTAATATTGAAACCC-3’ (Sequence Listing SEQ ID No: 1) and the reverse primer: 5’-GGGTTTCAATATTAGCAGGCCCCACCG-3’ (Sequence Listing SEQ ID No: 2) were selected. The single-point mutation of serine at position 188 was carried out using PCR technology. The PCR experimental procedure and the protein expression experimental procedure refer to Patent CN202311261575.4 and CN202410908168.6. The specific construction method is as follows: PCR conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 6 min, for a total of 25 cycles, and then extension at 72°C for 10 min. After PCR, 1 μL of DpnI was added and digested at 37°C for 1 h. Then, 10 μL was taken and transferred into 50 μL of E. coli DH5α competent cells by heat shock method, and then single clone strains were picked for sequencing verification. The successfully sequenced target plasmid was transferred into the E. coli expression strain BL21-DE3, and spread on an LB plate containing kanamycin, and cultured for 12 h. Single clone strains were picked into an LB (containing 50 μg / mL kanamycin) medium and cultured overnight at 37°C. Transferred to a TB (containing 50 μg / mL kanamycin) medium at an inoculation amount of 1% and cultured at 37°C for 4 h. After adding 0.5 mM IPTG for induction, continued to culture at 30°C for 18 h. Finally, the cells were collected by centrifugation at 4°C and 6000 rpm. The collected cell pellet was resuspended in phosphate buffer (10 mM, pH = 7.0), and the cells were lysed using an ultrasonic disruptor. Then, centrifuged at 10000 rpm and 4°C for 20 min, and the supernatant was collected as the crude enzyme solution of ChKRED20-M4C4.

[0043] Example 2

[0044] Construction of mutant ChKRED20-M4C3:

[0045] Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer: 5’-TGGGGCCTGCTGGCATTGAAACCCCGCTGTTGGAA-3’ (Sequence Listing SEQ ID No: 3) and the reverse primer: 5’-TTCCAACAGCGGGGTTTCAATGCCAGCAGGCCCCA-3’ (Sequence Listing SEQ ID No: 4) were selected. The single-point mutation of serine at position 188 was carried out using PCR technology. The specific construction method was as follows: PCR conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 6 min, for a total of 25 cycles, and then extension at 72°C for 10 min. After the PCR was completed, 1 μL of DpnI was added and digested at 37°C for 1 h. Then, 10 μL was taken and transformed into 50 μL of E. coli DH5α competent cells by heat shock method, and then single clone strains were picked for sequencing verification. The target plasmid with successful sequencing above was transferred into the E. coli expression strain BL21-DE3, spread on an LB plate containing kanamycin, and cultured for 12 h. Single clone strains were picked into an LB (containing 50 μg / mL kanamycin) medium and cultured overnight at 37°C. They were transferred to a TB (containing 50 μg / mL kanamycin) medium at an inoculation amount of 1% and cultured at 37°C for 4 h. After adding 0.5 mM IPTG for induction, the culture was continued at 30°C for 18 h. Finally, the cells were collected by centrifugation at 4°C and 6000 rpm. The collected cell pellet was resuspended in phosphate buffer (10 mM, pH = 7.0), and the cells were lysed using an ultrasonic disruptor. Then, centrifugation was carried out at 10000 rpm and 4°C for 20 min, and the supernatant was collected as the crude enzyme solution of ChKRED20-M4C3.

[0046] Example 3

[0047] Construction of mutant ChKRED20-M4C6:

[0048] Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, the forward primer: 5’-AATGCGGTGGGGCCTGCTACCATTGAAACCCC-3’ (Sequence Listing SEQ ID No: 5) and the reverse primer: 5’-GGGGTTTCAATGGTAGCAGGCCCCACCGCATT-3’ (Sequence Listing SEQ ID No: 6) were selected. The single-point mutation of serine at position 188 was carried out by PCR technology. The specific construction method is as follows: PCR conditions: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 6 min, for a total of 25 cycles, and then extension at 72°C for 10 min. After the PCR was completed, 1 μL of DpnI was added and digested at 37°C for 1 h. Then, 10 μL was taken and transformed into 50 μL of E. coli DH5α competent cells by heat shock method, and then single clone strains were picked for sequencing verification. The target plasmid with successful sequencing above was transferred into the E. coli expression strain BL21-DE3, spread on an LB plate containing kanamycin, and cultured for 12 h. Single clone strains were picked into an LB (containing 50 μg / mL kanamycin) medium and cultured overnight at 37°C. Transferred with an inoculation amount of 1% to a TB (containing 50 μg / mL kanamycin) medium and cultured at 37°C for 4 h. After adding 0.5 mM IPTG for induction, it was continued to be cultured at 30°C for 18 h. Finally, the cells were collected by centrifugation at 4°C and 6000 rpm. The collected cell pellets were resuspended in phosphate buffer (10 mM, pH = 7.0), and the cells were lysed using an ultrasonic disruptor. Then, centrifuged at 10000 rpm and 4°C for 20 min, and the supernatant was collected as the crude enzyme solution of ChKRED20-M4C6.

[0049] Example 4

[0050] The synthetic route of 1b with the configuration of (1S,2S) is as follows:

[0051]

[0052] Synthesis method: The substrate compound 1a (0.5 mg) and the crude enzyme solution of ChKRED20-M4C4 (200 μL) were dispersed in PBS buffer (700 μL, 50 mmol / L, pH = 7.9) containing NAD+ (2 mM), 100 μL of isopropanol was added, and then reacted at 37°C for 12 h to obtain (1S,2S)-1b.

[0053] Figure 1 is the 1H NMR spectrum of the racemate 1b, which proves the successful synthesis of the substance with the structure shown in (1S,2S)-1b.

[0054] Figure 2It is the liquid chromatogram of the catalytic synthesis of (1S,2S)-1b by ChKRED20-M4C4. Liquid chromatography conditions: IH chiral column, n-hexane: isopropanol = 90:10, flow rate = 1 mL / min, temperature = 30 °C, detection wavelength = 210 nm.

[0055] The synthesis methods of other similar compounds 2b - 10b and 1d - 4d are the same as those in Example 4. The specific data (yield, enantioselectivity, and diastereoselectivity) are as Figure 3 .

[0056] Example 5

[0057] The synthetic route of 11b with the configuration of (1S,2S) is as follows:

[0058]

[0059] Synthesis method: Disperse the substrate compound 11a (0.5 mg) and the crude enzyme solution of ChKRED20-M4C6 (200 μL) in PBS buffer containing NAD+ (2 mM) (700 μL, 50 mmol / L, pH = 7.9), add 100 μL of isopropanol, and then react at 37 °C for 12 h to obtain (1S,2S)-11b.

[0060] Figure 4 It is the 1H NMR spectrum of the racemate 11b, which proves the successful synthesis of the substance with the structure of (1S,2S)-11b.

[0061] Figure 5 It is the liquid chromatogram of the catalytic synthesis of (1S,2S)-1b by ChKRED20-M4C6. Liquid chromatography conditions: ADH chiral column, n-hexane: isopropanol = 90:10, flow rate = 1 mL / min, temperature = 30 °C, detection wavelength = 210 nm.

[0062] Example 6

[0063] The synthetic route of 12b with the configuration of (1S,2R) is as follows:

[0064]

[0065] Synthesis method: Substrate compound 12a (0.5 mg) and crude ChKRED20-M4C3 enzyme solution (200 μL) were dispersed in PBS buffer (700 μL, 50 mmol / L, pH = 7.9) containing NAD+ (2 mM), 100 μL of isopropanol was added, and then the reaction was carried out at 37 °C for 12 h to obtain (1S,2R)-12b. Scale-up reaction conditions: Weigh 100 mg of substrate 12a, dissolve it in 1 mL of DMSO, add 30 mL of PBS, 50 mg of NAD+, and 4 mL of isopropanol to a 100 mL conical flask. Subsequently, transfer the DMSO solution containing the substrate to the conical flask. Finally, add 5 mL of crude ChKRED20-M4C3 enzyme solution, react at 37 °C for 16 hours, extract the reaction solution with ethyl acetate and collect the organic phase, and purify it by vacuum distillation to finally obtain 57 mg of (1S,2R)-12b. The product morphology is shown in Figure 6 .

[0066] Figure 7 is the 1H NMR spectrum of racemic 12b, which proves the successful synthesis of the substance with the structure shown in (1S,2R)-12b.

[0067] Figure 8 is the liquid phase spectrum of the catalytic synthesis of (1S,2R)-12b by ChKRED20-M4C3. Liquid phase conditions: OD-3 chiral column, n-hexane:isopropanol = 96:4, flow rate = 1 mL / min, temperature = 30 °C, detection wavelength = 210 nm.

[0068] On the basis of the synthesis method in Example 4, the catalyst ChKRED20-M4C4 was replaced with ChKRED20-M4A1, ChKRED20-M3C4, ChKRED20-M3C1, ChKRED20-M2, and WT respectively to form Comparative Examples 1-5. ChKRED20-M3C4 also comes from Patent CN202410908168.6. Starting from the amino acid sequence of the carbonyl reductase mutant strain ChKRED20-M2, tyrosine at the 188th position was mutated to asparagine, and this mutant strain was named ChKRED20-M3C4. ChKRED20-M2 (Q97L / S153L) comes from Patent CN202311261575.4, and glutamine at the 97th position and serine at the 153rd position were both mutated to leucine. ChKRED20-M3C1 (Q97L / S153L / Y188S) comes from Patent CN202311261575.4, glutamine at the 97th position and serine at the 153rd position were both mutated to leucine, and the amino acid at the 188th position was mutated to serine. WT refers to wild-type ChKRED20. The detection results of the catalysts used in Comparative Examples 1-5 and the chiral values and yields of the synthesized products are shown in Table 1.

[0069] Table 1 Detection Results of Chiral Values and Yields

[0070] mutant yield [[ID= ​ ​ ​ 99 >99:1 98:2 ​ ​ 90 >99:1 64:36 ​ ​ 0 -- -- ​ ​ 0 -- -- ​ ​ 0 -- -- ​ ​ 0 -- --

[0071] As can be seen from Table 1, wild-type ChKRED20, ChKRED20-M2, ChKRED20-M3C1, and ChKRED20-M3C4 cannot catalyze the reduction of reactant 1a because the steric hindrance of the active cavity is large, which hinders the entry of the substrate into the enzyme activity. Although ChKRED20-M4A1 can synthesize 1b, its diastereoselectivity is poor because the volume of the enzyme's active cavity is too large, resulting in multiple binding modes of the substrate in the enzyme's active cavity. The mutant strain ChKRED20-M4C4 designed in the present invention overcomes the limitations of the above mutants because ChKRED20-M4C4 has a suitable active cavity volume, allowing the substrate to enter the active cavity and have a single binding conformation with the lowest energy in the enzyme's active cavity.

[0072] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent changes and modifications within the scope of the technical solution of the present invention by using the disclosed technical content above. However, as long as it does not depart from the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds, characterized in that, Using the mutant M4A1 of carbonyl reductase ChKRED20, abbreviated as ChKRED20-M4A1, as the parent, serine at position 188 was mutated to any one of asparagine, glycine or threonine to obtain a carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds.

2. The carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds according to claim 1, wherein Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, selecting the forward primer shown in SEQ ID No: 1 in the sequence listing and the reverse primer shown in SEQ ID No: 2 in the sequence listing, and using the PCR technique to perform a single-point mutation of serine at position 188 to asparagine to obtain the mutant ChKRED20-M4C4.

3. The carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds as described in claim 1, characterized in that, Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, selecting the forward primer shown in SEQ ID No: 3 in the sequence listing and the reverse primer shown in SEQ ID No: 4 in the sequence listing, and using the PCR technique to perform a single-point mutation of serine at position 188 to glycine to obtain the mutant ChKRED20-M4C3.

4. The carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds according to claim 1, characterized in that, Using the gene sequence of ChKRED20-M4A1 as the DNA template sequence, selecting the forward primer shown in SEQ ID No: 5 in the sequence listing and the reverse primer shown in SEQ ID No: 6 in the sequence listing, and using the PCR technique to perform a single-point mutation of serine at position 188 to threonine to obtain the mutant ChKRED20-M4C6.

5. Use of a carbonyl reductase mutant as described in any one of claims 1-4, characterized in that, It is used as a catalyst to catalyze the synthesis of chiral alcohol sulfone compounds.

6. Use of the carbonyl reductase mutant according to claim 5, characterized in that, The method for synthesizing chiral alcohol sulfone compounds is as follows: Disperse the substrate compound and the carbonyl reductase mutant for synthesizing chiral alcohol sulfone compounds in a PBS buffer containing β-nicotinamide adenine dinucleotide, add isopropanol, and then react at 35-40 °C for 10-16 h to obtain chiral alcohol sulfone compounds.

7. Use of the carbonyl reductase mutant according to claim 6, characterized in that, The mutant ChKRED20-M4C4 is used to catalyze the synthesis of any one of the following chiral alcohol sulfone compounds: In the formula, R1 is one of -H, -F, -Cl, -Me, R2 is one of -H, -F, -Cl, -Me, R3 is -CH3 or -Cl, and R4 is one of -CH2CH3, cyclopropyl, -N(CH3)2.

8. Use of the carbonyl reductase mutant according to claim 6, characterized in that, The mutant ChKRED20-M4C6 is used to catalyze the synthesis of the following compound:

9. Use of the carbonyl reductase mutant according to claim 6, characterized in that, The mutant ChKRED20-M4C3 is used to catalyze the synthesis of the following compound:

Images