Ketoreductase mutant and application thereof in preparation of (R)-2-hydroxy-3, 3-dimethylbutyric acid

Abstract

The invention discloses a ketoreductase mutant and an application of the ketoreductase mutant in preparation of (R)-2-hydroxy-3, 3-dimethyl butyric acid. The ketoreductase mutant disclosed by the invention can convert trimethylpyruvic acid into (R)-2-hydroxy-3, 3-dimethylbutyric acid, the conversion rate is greater than or equal to 85%, the chiral purity of the product is greater than or equal to 99%, and the method disclosed by the invention is simple to operate and high in yield, and can be used for industrial production.

Description

Technical field:

[0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a ketoreductase mutant and application thereof in preparing (R)-2-hydroxy-3,3-dimethylbutyric acid. Background technology:

[0002] (R)-2-Hydroxy-3,3-dimethylbutyric acid, with the structural formula shown in I and CAS number 22146-57-2, is an important pharmaceutical intermediate that can be used to synthesize thrombin inhibitor drug molecules for the treatment of thrombotic cardiovascular and cerebrovascular diseases. In the future, this compound can be used to prepare more new drug compounds.

[0003]

[0004] Patent US7109004B2 discloses a preparation route of compound I as shown in Scheme 1. The route uses 3,3-dimethylbutyric acid ethyl ester as a starting material, undergoes a reduction reaction under the action of KRED1001 to obtain (R)-2-hydroxy-3,3-dimethylbutyric acid ethyl ester, and hydrolyzes the ester group under alkaline conditions to obtain (R)-2-hydroxy-3,3-dimethylbutyric acid. The total yield of the two-step reaction is about 80%.

[0005]

[0006] Patent CN1497046A discloses a ketoacid reductase derived from Leuconostoc mesenteroides subsp. glucan strain ATCC 17072, which can directly reduce trimethylpyruvate to (R)-2-hydroxy-3,3-dimethylbutyrate, as shown in Scheme 2. However, the activity of the ketoacid reductase is low.

[0007]

[0008] Therefore, we need to construct a new ketoreductase and use it for the preparation of (R)-2-hydroxy-3,3-dimethylbutyrate. Summary of the invention:

[0009] The object of the present invention is to provide a ketoreductase mutant for preparing (R)-2-hydroxy-3,3-dimethylbutyrate in view of the deficiencies of the prior art.

[0010] On the one hand, the present invention provides a novel ketoreductase mutant, which is based on the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO.1 as a reference sequence, and single-point or multi-point mutations occur at positions 84, 94, 102, 131, 152, 180, 194, 201, 203, and 244. Among them, the valine at position 84 is mutated to leucine, the alanine at position 94 is mutated to glycine, the threonine at position 102 is mutated to serine, the asparagine at position 131 is mutated to methionine, the threonine at position 152 is mutated to serine, the aspartic acid at position 180 is mutated to asparagine, the proline at position 194 is mutated to tyrosine, the glycine at position 201 is mutated to alanine, the glutamic acid at position 203 is mutated to aspartic acid, and the valine at position 244 is mutated to leucine.

[0011] Furthermore, the wild-type ketoreductase is derived from Lentilactobacillus kefiri, the wild-type template NCBI accession number is WP_054768785.1, and the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0012] Furthermore, the amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO.3, 5, and 7.

[0013] Furthermore, the nucleotide sequence of the ketoreductase mutant is shown in SEQ ID NO.4, 6, and 8.

[0014] Furthermore, the ketoreductase mutant is expressed in E. coli BL(21)DE3.

[0015] Furthermore, the expression vector of the ketoreductase mutant is pET28a, but is not limited to the pET series.

[0016] In another aspect, the present invention provides a method for preparing (R)-2-hydroxy-3,3-dimethylbutyric acid using a ketoreductase mutant. The method uses trimethylpyruvic acid as a substrate and converts it into (R)-2-hydroxy-3,3-dimethylbutyric acid under the catalytic action of the ketoreductase mutant.

[0017] Furthermore, the ketoreductase mutant participates in the catalytic reaction in the form of ketoreductase enzyme powder, ketoreductase enzyme solution, ketoreductase homogenate solution, ketoreductase lyophilized powder, cells containing ketoreductase, etc., preferably ketoreductase homogenate solution and lyophilized powder.

[0018] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When cells containing ketoreductase are used, a small amount of coenzymes are contained in the cells, and the coenzymes may not be added at this time. In some cases, the prepared ketoreductase enzyme powder also contains a small amount of coenzymes, and the coenzymes may not be added at this time. However, coenzymes can also be added to the reaction system to further promote the reaction. When coenzymes are added to the reaction system to promote the reaction, the coenzymes are selected from NAD + , NADH, NADP + , NADPH or a combination thereof, preferably NADPH and NADP + .

[0019] Furthermore, the concentration of the added coenzyme is 0.02 to 0.4 g / L, preferably 0.04 to 0.2 g / L.

[0020] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0021] Furthermore, a hydrogen donor needs to be added in the reaction, and the hydrogen donor is selected from methanol, ethanol, isopropanol, and glucose, preferably glucose.

[0022] Furthermore, the substrate concentration is 10 to 300 g / L, preferably 10 to 200 g / L.

[0023] Furthermore, the reaction time is 1 h to 48 h, preferably 12 h to 24 h.

[0024] Furthermore, the reaction needs to be controlled at a pH of 6.0 to 8.0, preferably a pH of 6.5 to 7.5.

[0025] Furthermore, the reaction temperature is 20°C to 50°C, preferably 30°C to 40°C.

[0026] The beneficial effect of the present invention is that the present invention provides a new ketoreductase mutant, which can efficiently catalyze the conversion of trimethylpyruvate into (R)-2-hydroxy-3,3-dimethylbutyrate, with a conversion rate of ≥85% and a product chiral purity of ≥99%. The method is simple to operate and has high atom economy, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Electropherogram of wild-type KRED.

[0028] Figure 2 Protein expression profile of wild-type KRED. DETAILED DESCRIPTION

[0029] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0030] Example 1 Cloning and Construction of Wild-Type Ketoreductase

[0031] The KRED gene was amplified by designing upstream and downstream primers. The upstream primer was 5'-CGATCGCATATGACTGATCGTTTAAAA-3', and the underlined part was the restriction endonuclease NdeⅠ site; the downstream primer was 5'-ATGCTAGGAATTCTTATTGAGCA GTGTATCCACCATCGAC-3', and the underlined part was the restriction endonuclease EcoRⅠ site.

[0032] Prime STAR polymerase was used to amplify the target gene KRED with the participation of upstream and downstream primers; the PCR reaction conditions were: 98℃ pre-denaturation for 2min, each cycle included 95℃ denaturation for 25s, 60℃ annealing for 30s, 72℃ extension for 2min, a total of 30 cycles; and finally 72℃ derivatization for 5min. After the reaction was completed, the PCR product was detected on a 1% agarose gel. The results are shown in Figure 1 The length of the nucleic acid is consistent with the size published in the PDB database (759 bp), and its nucleotide sequence is shown in SEQ ID No: 2. The product was purified using a kit, and the purified product was double-digested with restriction endonucleases NdeⅠ and EcoRⅠ, and then the plasmid pET-28a was double-digested with the same endonucleases, and the cut gene and plasmid fragments were connected by T4 DNA ligase enzyme. After the ligation product was purified again, it was directly transformed into Escherichia coli DH5α competent cells, incubated at 37°C for 1 hour, and then directly coated on an LB plate containing 2 5μg / ml kanamycin resistance to obtain a recombinant plasmid named pET28a-KRED.

[0033] Example 2 Protein expression of wild-type KRED

[0034] The constructed recombinant plasmid pET28a-KRED was transformed into BL21(DE3) competent cells by chemical transformation and cultured in a cell culture medium containing Kan + Incubate the cells on LB plates containing KRED overnight at 37°C. Select positive monoclonal cells to obtain genetically engineered strains of ketoreductase that can be induced to express. Inoculate BL21 (DE3) cells containing KRED gene on plates containing Kan +In a resistant LB test tube, culture at 37°C overnight to obtain a primary seed culture solution. The seed culture solution was transferred to a 2YT liquid culture medium containing resistance at a 1% inoculation ratio, placed in a shaker, and cultured at 37°C and 200rpm for 3-5h. When the OD reached 0.6-0.8, the temperature was lowered to 20°C, and IPTG was added with the concentration controlled at 0.1mM for overnight induction of expression. The fermentation broth was centrifuged and the bacteria were collected. The collected bacteria were dissolved with 20mM phosphate buffer (pH7.5), and the cells were broken by ultrasound. After centrifugation again at 12000rpm for 10min, the supernatant obtained was the KRED protein. The expression of the protein can be seen by electrophoresis, see attached. Figure 2 .

[0035] Example 3 Construction of mutants

[0036] According to the simulated structure of KRED, sites 84, 94, 102, 131, 152, 180, 194, 201, 203, and 244 were mutated respectively. Mutation primers were designed using Primer 5 software, and mutants were obtained using whole plasmid PCR. The specific primer designs are shown in Table 1.

[0037] Table 1 Primer sequences for site-directed mutagenesis

[0038]

[0039]

[0040] The underlined sequences in Table 1 are mutation sites. Primer STAR max DNA polymerase was used with the participation of forward and reverse saturation primers and the recombinant plasmid pET28a-KRED as a template to perform multi-fragment overlapping PCR. The PCR reaction program is shown in Table 2.

[0041] Table 2 Full plasmid amplification program

[0042]

[0043] The obtained full plasmid mutant fragment gene was transferred into BL21 (DE3) competent cells and spread on a plate containing 30ug / mL Kan + The cells were cultured overnight at 37°C on a resistant LB plate, and a single clone was selected for sequencing to identify whether the site was mutated. After obtaining mutants at a single point, the activity was tested separately, and the mutants with improved activity were mutated again, and the mutation method was the same as above.

[0044] Example 4 Screening of the best mutants

[0045] The wild-type KRED and the monoclonal cells obtained after saturation mutation were picked up and cultured in 96-well plates, each containing 100uL of LB medium. After culturing at 37°C for 5h, 50ul / well of seed solution was transferred to a 96 deep-well plate for culture, each containing 1mL of 2YT medium. After culturing at 37°C for 5h, 10uL of IPTG was transferred to each well for induction culture at 25°C. After induction overnight, the cells were collected by centrifugation at 4000rpm for 30min for reaction screening.

[0046] The reaction was carried out in a deep 96-well plate. Trimethylpyruvate was added to each well. The reaction system was in 500uL. After shaking at 37°C for 12 hours, the reaction solution was analyzed by TLC spot plate analysis. The primary screening positive mutants Mut 1 (V84L / A94G / T102S), Mut 2 (V84L / A94G / T102S / N131M / T152S / D180N), and Mut3 (V84L / A94G / T102S / N131M / T152S / D180N / P194Y / G201A / E203D / V244L) were determined based on the reduction in trimethylpyruvate.

[0047] Example 5 Optimization of reaction conditions - selection of enzyme form

[0048] In order to optimize the effect of parameters on product synthesis during the transformation process, the morphology of the enzyme during the reaction of mutants Mut1, Mut2, and Mut3 was optimized respectively. The morphological parameters of the enzyme were set as lyophilized powder, cell homogenate, and cells. The reaction was carried out for 12 hours. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 3. The reaction results showed that the transformation effect was better when the enzyme was in the form of lyophilized powder and cell homogenate.

[0049] Table 3 Morphological optimization results of different enzymes

[0050]

[0051] Example 6 Optimization of reaction conditions---Selection of coenzyme

[0052] In order to optimize the effect of parameters on product synthesis during the conversion process, the coenzymes in the reaction process of mutants Mut1, Mut2, and Mut3 were optimized respectively. The coenzyme parameters were set to NAD, NADP, NADH, and NADPH. The reaction was carried out for 12 hours. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 4. The reaction results showed that the conversion effect was better when NADP and NADPH were used as coenzymes.

[0053] Table 4 Optimization results of different coenzymes

[0054]

[0055] Example 7 Optimization of reaction conditions - selection of hydrogen donor

[0056] In order to optimize the effect of parameters on product synthesis during the conversion process, the hydrogen donors in the reaction process of mutants Mut1, Mut2, and Mut3 were optimized respectively. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 5. The reaction results show that the conversion effect is better when isopropanol and glucose are selected as hydrogen donors.

[0057] Table 5 Optimization results of different hydrogen donors

[0058]

[0059] Example 8 Optimization of reaction conditions --- selection of temperature

[0060] In order to optimize the effect of parameters on product synthesis during the transformation process, the temperature during the reaction of mutants Mut1, Mut2, and Mut3 was optimized. The temperature was set in the range of 20-50°C and the reaction was carried out for 12 hours. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 6. The reaction results show that the temperature is 30-40°C and the transformation effect is better.

[0061] Table 6 Optimization results at different temperatures

[0062]

[0063] Example 9 Optimization of reaction conditions---Selection of pH

[0064] In order to optimize the effect of parameters on product synthesis during the transformation process, the pH during the reaction of mutants Mut1, Mut2, and Mut3 was optimized, and the pH was set between 6.0 and 8.0. The reaction was carried out for 12 hours. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 7. The reaction results showed that the transformation effect was better when the pH was 6.5-7.5.

[0065] Table 7 Optimization results of different pH values

[0066]

[0067] Example 10 Optimization of reaction conditions---Increasing substrate concentration

[0068] In order to optimize the effect of the conversion process on product synthesis, the substrate concentrations of the mutants Mut1, Mut2, and Mut3 during the reaction were optimized, and the substrate concentrations were set between 10 and 300 g / L. After the same operation, the synthesized products were finally analyzed. The specific data are shown in Table 8. The reaction results showed that when the substrate concentration was <200 g / L, the conversion rate was not much different from that of 10 to 100 g / L, and when the substrate concentration was >200 g / L, the conversion rate decreased significantly. Therefore, the optimal substrate concentration was 10 to 200 g / L, the conversion rate was >85%, and the product ee value was >99%.

[0069] Table 8 Optimization results of different substrate concentrations

[0070]

Claims

1. A ketoreductase mutant, characterized in that Taking the wild-type ketoreductase amino acid sequence shown in SEQ ID NO.1 as the reference sequence, single or multiple mutations occur at positions 84, 94, 102, 131, 152, 180, 194, 201, 203, and 244. Among them, the valine at position 84 is mutated to leucine, the alanine at position 94 is mutated to glycine, the threonine at position 102 is mutated to serine, the asparagine at position 131 is mutated to methionine, the threonine at position 152 is mutated to serine, the aspartic acid at position 180 is mutated to asparagine, the proline at position 194 is mutated to tyrosine, the glycine at position 201 is mutated to alanine, the glutamic acid at position 203 is mutated to aspartic acid, and the valine at position 244 is mutated to leucine.

2. The ketoreductase mutant according to claim 1, characterized in that The amino acid sequences of the ketoreductase mutants are shown in SEQ ID NOs. 3, 5, and 7.

3. The ketoreductase mutant according to claim 1, characterized in that The nucleotide sequences of the ketoreductase mutants are shown in SEQ ID NOs. 4, 6 and 8.

4. The ketoreductase mutant according to claim 1, characterized in that The ketoreductase mutant was expressed in E. coli BL(21)DE3.

5. A method for preparing (R)-2-hydroxy-3,3-dimethylbutyric acid, characterized in that: Trimethylpyruvate is converted into (R)-2-hydroxy-3,3-dimethylbutyrate under the catalytic action of a ketoreductase mutant, wherein the ketoreductase mutant is selected from the ketoreductase mutant in claim 1.

6. The preparation method according to claim 5, characterized in that: The ketoreductase mutant participates in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenized solution, lyophilized powder, cells containing the enzyme, and the like.

7. The preparation method according to claim 5, characterized in that: The coenzyme is selected from NAD + , NADH, NADP + , NADPH or a combination thereof.

8. The preparation method according to claim 5, characterized in that: The substrate concentration is 10-300 g / L.

9. The preparation method according to claim 5, characterized in that: The reaction temperature is 20°C to 50°C.

10. The preparation method according to claim 5, characterized in that: The reaction needs to be controlled at pH 6.0-8.0.

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