Carbonyl reductase mutant as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of enzyme engineering, and provides a carbonyl reductase mutant as well as a preparation method and application thereof, the carbonyl reductase mutant is used for asymmetrically and specifically catalyzing a prochiral carbonyl compound to generate corresponding chiral alcohol; the carbonyl reductase mutant takes the sequence of the carbonyl reductase polypeptide as a starting sequence, and compared with the sequence of the carbonyl reductase polypeptide, the carbonyl reductase mutant contains one or more residue differences; the amino acid sequence of the carbonyl reductase polypeptide is as shown in SEQ ID NO. 2 in the sequence table. The enzyme activity of the carbonyl reductase mutant obtained by carrying out site-directed mutagenesis on the carbonyl reductase is far higher than that of the carbonyl reductase which is not mutated; the carbonyl reductase mutant can be used for specifically catalyzing a prochiral carbonyl compound into a chiral product of a required Ritlecitinib medical intermediate, so that the carbonyl reductase mutant has a wide application value and a very strong practical value in the synthesis application of a medicament Ritlecitinib for treating alopecia areata.

Description

TECHNICAL FIELD

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

[0002] Chiral alcohol is an important intermediate for synthesizing chiral drugs, and asymmetric reduction of carbonyl compounds by biological catalysis to produce chiral alcohol is one of important methods for preparing chiral alcohol. Biological catalysis usually takes carbonyl reductase (or ketoreductase)-producing microorganisms or recombined enzymes coupled with a coenzyme regeneration system as a biological catalyst. Since the recombined enzyme has almost no side reaction in the reaction, most of the large-scale industrial production currently adopts the recombined enzyme. However, with the rapid growth of the demand for customized biological catalysts, the existing basic enzyme library cannot meet the needs in quantity and quality. Protein engineering technology for molecular modification of enzymes is a recognized favorable tool for solving enzyme limitations, and can quickly obtain enzyme mutants with improved enzymatic properties in a target-oriented manner to meet the requirements of industrial production processes.

[0003] Ritlecitinib is a specific JAK3 / TEC inhibitor with high selectivity for tyrosine kinase members expressed in the TEC kinase family and Janus kinase 3 (JAK3), which can block the signal molecules and immune cell activity leading to alopecia areata, thereby inhibiting the immune system to kill hair follicle cells to achieve the purpose of treating alopecia areata. The chemical preparation of the Ritlecitinib pharmaceutical intermediate has been reported, but due to the complexity of the chemical reaction process, the need to use expensive transition metals, harsh reaction conditions, tedious separation and purification process, poor atom economy, and environmental unfriendliness, etc., therefore, there is an urgent need for a biological catalyst with excellent performance to prepare the Ritlecitinib pharmaceutical intermediate. SUMMARY

[0004] The present application aims to provide a carbonyl reductase mutant, which aims to solve the problems presented in the background.

[0005] To solve the above problems, the present application is achieved as follows: the carbonyl reductase mutant is used for asymmetric specific catalysis of prochiral carbonyl compound to generate corresponding chiral alcohol; the carbonyl reductase mutant takes the sequence of the carbonyl reductase polypeptide as a starting sequence, and contains one or more residue differences selected from 7S, 9G, 23V, 27L, 27S, 60I, 64V, 71R, 87L, 89M, 93A, 93S, 93I, 94R, 95M, 96E, 96F, 96R, 97A, 97F, 97R, 97V, 98V, 127R, 131S, 144V, 145L, 152G, 188A, 188F, 188V, 188Y, 188M, 194F, 194L, 194V, 196F, 197A, 200R, 202A, 202L, 208R, 216R, 245I, 249F, 249G, 249Y compared with the sequence of the carbonyl reductase polypeptide; the amino acid sequence of the carbonyl reductase polypeptide is shown in the sequence table SEQ ID NO. 2; the numerical part of the residue difference represents the position number of the amino acid residue in the starting sequence, and the letter part represents the amino acid type after mutation of the original amino acid residue at the corresponding position number.

[0006] Preferably, the nucleotide sequence of the carbonyl reductase polypeptide is shown in the sequence table SEQ ID NO. 1.

[0007] Preferably, the carbonyl reductase mutant contains one or more residue differences selected from 93A, 93S, 96R, 97A, 97F, 97R, 97V, 188V, 194V, 194F, 197A, 202A compared with the sequence of the carbonyl reductase polypeptide.

[0008] Another object of the present application is to provide a polynucleotide encoding the above-mentioned carbonyl reductase mutant.

[0009] Another object of the present application is to provide an expression vector containing the above-mentioned polynucleotide.

[0010] Another object of the present application is to provide a host cell containing the above-mentioned polynucleotide or the above-mentioned expression vector.

[0011] Another object of the present application is to provide a preparation method of the above-mentioned carbonyl reductase mutant, comprising the following steps:

[0012] According to the gene sequence of the carbonyl reductase polypeptide, a carbonyl reductase upstream primer, a carbonyl reductase upstream primer, a mutant upstream primer, and a mutant downstream primer are designed;

[0013] The plasmid of the wild-type carbonyl reductase polypeptide is used as a template, and the carbonyl reductase upstream primer, the carbonyl reductase upstream primer, the mutant upstream primer and the mutant downstream primer are used for PCR amplification of the site-directed mutant carbonyl reductase to obtain a PCR product;

[0014] The PCR product is purified and connected to an expression vector after double enzyme digestion, and then transformed into host cells to obtain a recombinant engineering bacterium;

[0015] The recombinant engineering bacterium is induced for expression to obtain the carbonyl reductase mutant.

[0016] Another object of the application is to provide one or more applications of the above-mentioned carbonyl reductase mutant in asymmetric specific catalysis of prochiral carbonyl compounds to generate corresponding chiral alcohols; here, multiple refers to a mixed enzyme of multiple carbonyl reductase mutants.

[0017] Preferably, the prochiral carbonyl compound has a structural general formula of formula I:

[0018] ;

[0019] Formula I

[0020] The chiral alcohol has a structural general formula of formula II:

[0021] ;

[0022] Formula II

[0023] In the formula, R1 and R2 are each independently selected from any one of H, optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxycarbonyl, arylalkoxycarbonyl, heteroarylalkoxycarbonyl and a protecting group.

[0024] Preferably, the prochiral carbonyl compound is ethyl (2R)-2-{[(tert-butoxy) carbonyl] amino}-5-oxohexanoate; and the chiral alcohol is used as a Ritlecitinib pharmaceutical intermediate.

[0025] Compared with the prior art, the application has the following advantages:

[0026] The carbonyl reductase mutant obtained by site-directed mutation of the carbonyl reductase has much higher enzyme activity than the carbonyl reductase without mutation; the carbonyl reductase mutant can be used for specific catalysis of prochiral carbonyl compounds into the desired chiral product of the Ritlecitinib pharmaceutical intermediate, and therefore has wide application value and strong practical value in the synthesis of the treatment of alopecia areata drug Ritlecitinib. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 This is the SDS-PAGE protein electrophoresis identification result provided in an embodiment of the present invention. In the figure, M: low molecular weight standard protein marker (TaKaRa); lanes 1 and 3 are the control pET-28a(+) and SDR, respectively. 20 -L197A whole cell culture; lanes 2 and 4 are pET-28a(+) and SDR respectively. 20 The supernatant of the L197A sample protein. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents, raw materials, and equipment used in this invention are commercially available.

[0030] In one embodiment of the present invention, a carbonyl reductase mutant is provided for asymmetric specific catalysis of a prochiral carbonyl compound to the corresponding chiral alcohol; the carbonyl reductase mutant uses the sequence of a carbonyl reductase polypeptide as a starting sequence and contains one or more residues selected from the following compared to the sequence of the carbonyl reductase polypeptide: 7S, 9G, 23V, 27L, 27S, 60I, 64V, 71R, 87L, 89M, 93A, 93S, 93I, 94R, 95M, 96E The amino acid sequences of the carbonyl reductase polypeptide are as shown in SEQ ID NO. 2 of the sequence listing. The numerical part of the residue difference represents the position number of the amino acid residue in the starting sequence, and the letter part represents the type of amino acid after mutation of the original amino acid residue at the corresponding position number (a single letter abbreviation of the amino acid).

[0031] These residue differences occur at residue positions that affect activity, stereoselectivity, substrate binding, expression, and their various combinations.

[0032] In a preferred embodiment of the present invention, the amino acid sequence of the carbonyl reductase polypeptide is shown in SEQ ID NO.2 of the sequence listing, and it is derived from Chryseobacterium, exhibiting high activity and high enantioselectivity.

[0033] In a preferred embodiment of the present invention, the carbonyl reductase mutant contains one or more residues selected from the group consisting of carbonyl reductase polypeptide that differ from the sequence of the latter: 93A, 93S, 96R, 97A, 97F, 97R, 97V, 188V, 194V, 194F, 197A, 202A.

[0034] In another embodiment of the present invention, a method for preparing the above-mentioned carbonyl reductase mutant is also provided, comprising the following steps:

[0035] S1. Based on the gene sequence of the carbonyl reductase polypeptide, design the upstream primer of carbonyl reductase, the upstream primer of carbonyl reductase, the mutant upstream primer, and the mutant downstream primer;

[0036] S2. Using the plasmid of wild-type carbonyl reductase polypeptide as a template, the site-directed mutated carbonyl reductase was amplified by PCR using the upstream primer of carbonyl reductase, the upstream primer of carbonyl reductase, the upstream primer of mutant, and the downstream primer of mutant to obtain the PCR product.

[0037] S3. The PCR product is purified, digested with two enzymes, ligated into an expression vector, and then transformed into a host cell to obtain recombinant engineered bacteria.

[0038] S4. The recombinant engineered bacteria are induced to express the carbonyl reductase mutant.

[0039] The expression vector can be pET-28a(+), but is not limited to it; the host cell can be E. coli BL21(DE3), but is not limited to it; the induction conditions are: use IPTG with a final concentration of 10-1000 μM for induction, and the induction temperature is 16-30℃.

[0040] In another embodiment of the present invention, an application of the above-mentioned carbonyl reductase mutant in the asymmetric specific catalysis of pre-chiral carbonyl compounds to generate corresponding chiral alcohols is also provided.

[0041] Specifically, the general structural formula of the prochiral carbonyl compound is Formula I:

[0042] ;

[0043] Formula I

[0044] The general structural formula of the chiral alcohol is Formula II:

[0045] ;

[0046] Formula II

[0047] In the formula, R1 and R2 are each independently selected from H, optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxycarbonyl, arylalkoxycarbonyl, heteroarylalkoxycarbonyl, and protecting group.

[0048] In a preferred embodiment of the present invention, the prochiral carbonyl compound is ethyl(2R)-2-{[(tert-butoxy)carbonyl]amino}-5-oxohexanoate. The aforementioned carbonyl reductase mutant can be used for asymmetric and specific catalysis of ethyl(2R)-2-{[(tert-butoxy)carbonyl]amino}-5-oxohexanoate to generate the Ritlecitinib pharmaceutical intermediate.

[0049] Under suitable reaction conditions, a prochiral carbonyl compound reacts with the aforementioned carbonyl reductase mutant in the presence of the cofactor NADPH or NADH to prepare a chiral alcohol.

[0050] The following embodiments are some specific implementation examples and application examples of the present invention in practical applications, but are not limited thereto.

[0051] Example 1: This example provides a method for cloning a carbonyl reductase gene and mutagenizing related sites, as detailed below:

[0052] S1, based on carbonyl reductase SDR 20 Primers were designed based on the gene sequence (nucleotide sequence shown in SEQ ID NO.1) (amino acid sequence as shown in SEQ ID NO.2, sequence listing):

[0053] SDR 20 -F: 5'-CGCGGATCCATGGGCATTCTGGATAATAAAGTGGC-3' (nucleotide sequence as shown in SEQ ID NO.3 of the sequence listing);

[0054] SDR 20 -R: 5'-GCCAAGCTTTTACACCGCGGTATAGCCGCCATCC-3' (nucleotide sequence as shown in SEQ ID NO.4 of the sequence listing);

[0055] SDR 20 -L197A-F: 5'-CATTGGCGGCGCGGCAGCGCTGACTGGTGATTATG-3' (nucleotide sequence as shown in SEQ ID NO.5 of the sequence listing);

[0056] SDR 20 -L197A-R: 5'-CATAATCACCAGTCAGCGCTGCCGCGCCGCCAATG-3' (nucleotide sequence as shown in SEQ ID NO. 6 of the sequence listing);

[0057] S2, with wild-type carbonyl reductase SDR 20 Using the plasmid as a template, the above primers were used to perform PCR amplification of the site-directed mutagenesis carbonyl reductase. The system is as follows:

[0058] The PCR procedure was as follows: pre-denaturation: 94℃, 5 min; PCR reaction: denaturation: 94℃, 30 s; annealing: Tm-5℃, 30 s; extension: 72℃, 30 s; repeat the denaturation-annealing-extension process for 30 cycles; final extension: 72℃ for 10 min. Storage: 4℃.

[0059] The first round of PCR system includes fragment 1 and fragment 2, as detailed below:

[0060] Fragment 1: 0.75 μL pET-28a(+)-SDR20 template, SDR 20 -F 0.75μL, SDR 20 -L197A-R 0.75μL, primeSTAR DNA polymerase 12.5μL, ddH2O 10.25μL;

[0061] Fragment 2: 0.75 μL of pET-28a(+)-SDR20 template, SDR 20 -R 0.75μL, SDR 20 -L197A-F 0.75μL, primeSTAR DNA polymerase 12.5μL, ddH2O 10.25μL.

[0062] The second round of PCR system is as follows:

[0063] The amount of fragment 1 added is calculated as three base sequence fragments. Fragment 2 is added in an equimolar ratio with fragment 1, and the total amount added is 3 μL. The amounts of other substances added are as follows: SDR 20 -F 3μL, SDR 20 -R 3μL, primeSTAR DNA polymerase 50μL, ddH2O to make up to 100μL.

[0064] The PCR procedure was as follows: pre-denaturation: 94℃, 5 min; PCR reaction: denaturation: 94℃, 30 s, annealing: Tm -5℃, 30 s, extension: 72℃, 30 s, with the denaturation-annealing-extension process repeated 30 times; final extension: 72℃, 10 min. Storage: 4℃. After PCR amplification, 2 μL of the PCR product was subjected to agarose gel electrophoresis, and the remaining product was stored at -20℃ for later use.

[0065] The PCR product was purified using a gel extraction kit and digested with BamHI and HindIII at 37°C for 8 h. It was then ligated with the pET-28a(+) expression vector that had been double-digested with BamHI and HindIII. 10 μL of the ligation product was transformed into Escherichia coli BL21(DE3). The transformation solution was plated on LB solid medium containing kanamycin sulfate (100 μg / mL) and incubated overnight at 37°C. Five single colonies were randomly selected and inoculated to extract plasmid DNA. After double digestion for verification, the samples were sent for sequencing to obtain the recombinant engineered bacteria.

[0066] Example 2: This example provides a method for expressing recombinant engineered bacteria and obtaining carbonyl reductase mutants, as detailed below:

[0067] The recombinant engineered bacteria successfully sequenced in Example 1 were streaked onto LB solid medium containing kanamycin sulfate (100 μg / mL) and incubated overnight at 37°C for activation. One recombinant bacterial strain was randomly selected and inoculated into LB liquid medium containing kanamycin sulfate (100 μg / mL) and incubated overnight at 37°C and 220 rpm. The inoculum was then transferred to 50 mL of LB liquid medium containing kanamycin sulfate (100 μg / mL) at a 1:100 inoculation ratio. When the bacterial density OD600 = 0.5-0.6, IPTG was added to a final concentration of 0.1 mM. The culture was then incubated at 25°C and 200 rpm for 10 h to obtain the induced product. Next, the induced product was centrifuged at 14,000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were resuspended in 50 mL of 100 mM potassium phosphate (pH=6.8) buffer. The cells were then disrupted using an ultrasonic disruptor (Sonics), followed by centrifugation at 14,000 rpm for 10 min at 4 °C. The supernatant was collected to obtain the crude recombinant protein. The crude recombinant protein was purified using a Ni-NTA (Shanghai Sangon Biotech) affinity column. The specific operation steps of the affinity column were performed according to the Shanghai Sangon Biotech product instructions. The carbonyl reductase mutant SDR was obtained. 20 -L197A, and finally the purity was identified by SDS-PAGE protein electrophoresis, the results are as follows: Figure 1 As shown, the protein concentration was determined using the BCA Protein Assay kit. This carbonyl reductase mutant SDR... 20 The sequence of -L197A is similar to that of the original carbonyl reductase SDR. 20In contrast, there is a difference in one residue: 197A, where the amino acid residue at position 197 in the sequence is mutated from the original L (Leu) to A (Ala).

[0068] Example 3: An application of the above-mentioned carbonyl reductase mutant is provided in the following example:

[0069] The carbonyl reductase SDR was determined by measuring the absorbance of the cofactor NADH at 340 nm. 20 and carbonyl reductase mutant SDR 20 -L197A enzyme activity. Specifically, a 10.0 mol / L NAD+ solution was prepared using potassium phosphate buffer at pH 6.8. + The stock solution was then diluted to a final concentration of 0.01 mol / L NAD. + The solution was added to a total volume of 300.0 μL of enzyme activity assay system, and 10% (v / v) of ethyl (2R)-2-{[(tert-butoxy)carbonyl]amino}-5-oxohexanoate was added as a substrate. Then, 10 μL of enzyme was mixed to obtain the reaction solution. The solution was incubated at 27 °C for different times, and the enzyme activity was measured at OD340.

[0070] Next, an equal volume of ethyl acetate was added to the above reaction solution and vortexed thoroughly to mix. The upper organic phase was extracted, and the extraction process was repeated three times. The solution was evaporated to dryness, resuspended in 500 μL of methanol, and then analyzed by high-performance liquid chromatography (HPLC). The detection and analysis conditions were as follows:

[0071] HPLC detection conditions: C18 (4.6 x 250 mm) column, mobile phase: methanol-0.1% glacial acetic acid, flow rate: 0.8 mL / min, detection wavelength: 254 nm, injection volume: 10 μL. At a substrate concentration of 10 mmol / L, it was compared with SDR... 20 After reacting with -L197A for 5 min, the experimental results showed that the substrate conversion rate was 73.49%, the product was in the R configuration, and the ee value was >85%.

[0072] Example 4: Using carbonyl reductase SDR 20 The sequence (amino acid sequence shown in SEQ ID NO.2 of the sequence listing) is the starting sequence. The carbonyl reductase mutant SDR is constructed according to the methods described in Examples 1-2 above. 20 -Q97A / M202A, SDR 20 -I93A / L194V, SDR 20 -E96R / Q97V, SDR 20 -L194F / Y188V, SDR 20 -L197A / Q97R, SDR 20 -M202A / Q97F, SDR 20-I93S / Y188V, SDR 20 -M202A, SDR 20 -Y188V, SDR 20 -L194F, SDR 20 -Q97A, SDR 20 -I93A / L197A, SDR 20 -E96A, SDR 20 -M202A / L197F, SDR 20 -E96A / L197A.

[0073] Among them, SDR 20 -Q97A / M202A sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 97A and 202A. Specifically, the amino acid residue at position 97 is mutated from Q (Gln) to A (Ala), and the amino acid residue at position 202 is mutated from M (Met) to A (Ala). SDR 20 -I93A / L194V sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 93A and 194V, meaning that the amino acid residue at position 93 in the sequence is mutated from the original I (Ile) to A (Ala), and the amino acid residue at position 194 is mutated from the original L (Leu) to V (Val); SDR 20 -E96R / Q97V sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 96R and 97V, meaning that the amino acid residue at position 96 in the sequence is mutated from the original E (Glu) to R (Arg), and the amino acid residue at position 97 is mutated from the original Q (Gln) to V (Val); SDR 20 -L194F / Y188V sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 194F and 188V, meaning that the amino acid residue at position 194 in the sequence is mutated from the original L (Leu) to F (Phe), and the amino acid residue at position 188 is mutated from the original Y (Tyr) to V (Val); SDR 20 -L197A / Q97R sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 197A and 97R, meaning that the amino acid residue at position 197 in the sequence is mutated from L (Leu) to A (Ala), and the amino acid residue at position 97 is mutated from Q (Gln) to R (Arg); SDR 20 -M202A / Q97F sequence and carbonyl reductase SDR 20In contrast, it contains two residue differences: 202A and 97F, meaning that the amino acid residue at position 202 in the sequence is mutated from the original M (Met) to A (Ala), and the amino acid residue at position 97 is mutated from the original Q (Gln) to F (Phe); SDR 20 -I93S / Y188V sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 193S and 188V, meaning that the amino acid residue at position 193 in the sequence is mutated from the original I (Ile) to S (Ser), and the amino acid residue at position 188 is mutated from the original Y (Tyr) to V (Val); SDR 20 -M202A sequence and carbonyl reductase SDR 20 In comparison, it contains one residue difference: 202A, meaning the 202nd amino acid residue in the sequence is mutated from the original M (Met) to A (Ala); SDR 20 -Y188V sequence and carbonyl reductase SDR 20 In comparison, it contains one residue difference: 188V, meaning the 188th amino acid residue in the sequence is mutated from the original Y (Tyr) to V (Val); SDR 20 -L194F sequence and carbonyl reductase SDR 20 In comparison, it contains one residue difference: 194F, meaning the 194th amino acid residue in the sequence is mutated from the original to F (Phe); SDR 20 -Q97A sequence and carbonyl reductase SDR 20 In comparison, it contains one residue difference: 97A, meaning the 97th amino acid residue in the sequence is mutated from the original Q (Gln) to A (Ala); SDR 20 -I93A / L197A sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 93A and 197A, meaning that the amino acid residue at position 93 in the sequence is mutated from the original I (Ile) to A (Ala), and the amino acid residue at position 197 is mutated from the original L (Leu) to A (Ala); SDR 20 -E96A sequence and carbonyl reductase SDR 20 In comparison, it contains one residue difference: 96A, meaning the 96th amino acid residue in the sequence is mutated from the original E (Glu) to A (Ala); SDR 20 -M202A / L197F sequence and carbonyl reductase SDR 20In contrast, it contains two residue differences: 202A and 197F, meaning that the amino acid residue at position 202 in the sequence is mutated from the original M (Met) to A (Ala), and the amino acid residue at position 197 is mutated from the original L (Leu) to F (Phe); SDR 20 -E96A / L197A sequence and carbonyl reductase SDR 20 In contrast, it contains two residue differences: 96A and 197A. That is, the amino acid residue at position 96 in the sequence is mutated from the original E (Glu) to A (Ala), and the amino acid residue at position 197 is mutated from the original L (Leu) to A (Ala).

[0074] The carbonyl reductase mutant SDR was subjected to the method provided in Example 3. 20 -Q97A / M202A, SDR 20 -I93A / L194V, SDR 20 -E96R / Q97V, SDR 20 -L194F / Y188V, SDR 20 -L197A / Q97R, SDR 20 -M202A / Q97F, SDR 20 -I93S / Y188V, SDR 20 -M202A, SDR 20 -Y188V, SDR 20 -L194F, SDR 20 -L194F and the original carbonyl reductase SDR 20 Performance tests were conducted, and the results are shown in Table 1.

[0075] In addition, the carbonyl reductase mutant SDR mentioned above 20 -Q97A, SDR 20 -L194F, SDR 20 -L197A / Q97R, SDR 20 -Q97A / M202A were mixed in an equal mass ratio to obtain mixed enzyme A; the above carbonyl reductase mutant SDR was then used. 20 -Y188V, SDR 20 -Q97A, SDR 20 -202A / Q97F, SDR 20 -I93A / L197A were mixed in an equal mass ratio to obtain mixed enzyme B; the above carbonyl reductase mutant SDR was then used. 20 -L197A, SDR 20 -E96A, SDR 20 -M202A / L197F, SDR 20-E96A / L197A were mixed in equal mass ratio to obtain mixed enzyme C; the performance of mixed enzyme A, mixed enzyme B and mixed enzyme C were tested according to the method provided in Example 3, and the results are shown in Table 1.

[0076] Table 1

[0077]

[0078] As can be seen from Table 1, the carbonyl reductase mutant obtained by site-directed mutagenesis of carbonyl reductase in the embodiments of the present invention has a much higher enzyme activity than the unmutated carbonyl reductase.

[0079] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is used for asymmetric specific catalysis of pre-chiral carbonyl compounds to generate the corresponding chiral alcohols; the carbonyl reductase mutant uses the sequence of the carbonyl reductase polypeptide as the starting sequence and contains one or more residues selected from the following compared to the sequence of the carbonyl reductase polypeptide: 7S, 9G, 23V, 27L, 27S, 60I, 64V, 71R, 87L, 89M, 93A, 93S, 93I, 94R, 95M, 96E, 96F, 96R The amino acid sequences of the carbonyl reductase polypeptide are as shown in SEQ ID NO. 2 of the sequence listing. The numerical part of the residue difference represents the position number of the amino acid residue in the starting sequence, and the letter part represents the type of amino acid after mutation of the original amino acid residue at the corresponding position number.

2. The carbonyl reductase mutant according to claim 1, characterized in that, The nucleotide sequence of the carbonyl reductase polypeptide is shown in SEQ ID NO.1 of the sequence listing.

3. The carbonyl reductase mutant according to claim 1, characterized in that, The carbonyl reductase mutant contains one or more residues selected from the following that differ from the sequence of the carbonyl reductase polypeptide: 93A, 93S, 96R, 97A, 97F, 97R, 97V, 188V, 194V, 194F, 197A, 202A.

4. A polynucleotide encoding a carbonyl reductase mutant according to any one of claims 1-3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the polynucleotide of claim 4 or the expression vector of claim 5.

7. The method for preparing the carbonyl reductase mutant according to any one of claims 1-3, characterized in that, Includes the following steps: Based on the gene sequence of the carbonyl reductase polypeptide, design upstream primers, upstream primers of carbonyl reductase, mutant upstream primers, and mutant downstream primers; Using a plasmid of wild-type carbonyl reductase polypeptide as a template, PCR amplification of site-directed mutated carbonyl reductase was performed using carbonyl reductase upstream primer, carbonyl reductase upstream primer, mutant upstream primer, and mutant downstream primer to obtain PCR products. The PCR product was purified, digested with two enzymes, ligated into an expression vector, and then transformed into a host cell to obtain recombinant engineered bacteria. The recombinant engineered bacteria were induced to express the carbonyl reductase mutant.

8. The use of one or more carbonyl reductase mutants as described in any one of claims 1-3 in the asymmetric specific catalysis of pre-chiral carbonyl compounds to generate corresponding chiral alcohols.

9. The application as described in claim 8, characterized in that, The general structural formula of the prochiral carbonyl compound is Formula I: ； Formula I The general structural formula of the chiral alcohol is Formula II: ； Formula II In the formula, R1 and R2 are each independently selected from H, optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, alkoxycarbonyl, arylalkoxycarbonyl, heteroarylalkoxycarbonyl, and protecting group.

10. The application as described in claim 9, characterized in that, The prochiral carbonyl compound is ethyl(2R)-2-{[(tert-butoxy)carbonyl]amino}-5-oxohexanoate; the chiral alcohol is used as a pharmaceutical intermediate for Ritlecitinib.

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