An improved ketoreductase and its application in the preparation of ezetimibe

Abstract

The improved ketoreductase of the present invention belongs to the technical field of biocatalytic synthesis. The ketoreductase mutant of the present invention has an amino acid sequence that is a mutated amino acid sequence of the amino acid sequence shown in SEQ ID NO:1. The mutation sites of the mutated amino acid sequence are: the 253rd amino acid V is mutated to F, or the 304th amino acid R is mutated to I, or both mutations occur simultaneously. The present invention provides a ketoreductase and its application, which improves the tolerance of the enzyme in organic solvents, effectively reduces the industrial production costs of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one and ezetimibe, and improves the product quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalytic synthesis. Specifically, it relates to an improved ketoreductase and a method for preparing the ezetimibe chiral intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxyvaleryl]-4-phenyl-1,3-oxazolidin-2-one and its application. Background Art

[0002] Ezetimibe, chemically named: (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-2-azetidinone, is the first cholesterol absorption inhibitor drug jointly developed by Schering-Plough Corporation and Merck and launched in 2002, with the trade name ezetrol. As an adjuvant therapy in addition to diet control, it can be used alone or in combination with HMG-CoA reductase inhibitors (statins) to treat primary (heterozygous familial or non-familial) hypercholesterolemia, and can reduce total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (Apo B).

[0003] Compound 2 ((4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxyvaleryl]-4-phenyl-1,3-oxazolidine (also known as oxazolidinone)-2-one, CAS: 189028-95-3) is a key chiral intermediate in the synthesis of ezetimibe API. Using chemical methods ( Figure 1 ) to synthesize Compound 2 involves asymmetric reduction in the step of reducing the ketone to a chiral hydroxyl group, and the catalysts and ligands used are expensive, resulting in high costs. Among the reported preparation processes, the most advantageous one is to use ketoreductase (KRED) ( Figure 2 ) to catalyze Compound 1 ((4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, CAS: 189028-93-1) for preparation, which can achieve high optical purity and conversion rate, and the cost of Compound 2 can be greatly reduced.

[0004] Amit Singh et al. discovered a strain Burkholderia cenocepacia that can catalyze the formation of Compound 2 from Compound 1. By optimizing the conditions, when the substrate feeding concentration is 0.71 g / L, the product formation rate is 54%, and e.e. > 99%. Liu Zhiqiang et al. (CN 104630243 B) discovered a kind of Burkholderia gladioli (Burkholderia gladioli) ZJB-12126 carbonyl reductase, the gene of this enzyme was heterologously expressed in Escherichia coli to obtain a recombinant strain. The recombinant cells were used as biocatalysts to prepare compound 2 using compound 1 as a substrate. At a substrate feeding concentration of 7.1 g / L, the product de > 99%, and the substrate conversion rate was 26.4% in 12 hours. Liu Zhiqiang et al. (CN 105039361 A) also discovered a carbonyl reductase from Rhodosporidium toruloides Rhodosporidium toruloides ) ZJB14212, the gene of this enzyme was heterologously expressed in Escherichia coli to obtain a recombinant strain. Similarly, the recombinant cells were used as biocatalysts to prepare compound 2 using compound 1 as a substrate. When the substrate concentration was 35 g / L, the substrate conversion rate was 91% and e.e. > 99%. Codexis Company (CN 102186972 B) used protein engineering technology to Lactobacillus kefir perform multiple rounds of modification on the carbonyl reductase in

[0005] and combined the beneficial mutations in each round to finally obtain an ideal carbonyl reductase mutant. Its activity and selectivity were greatly improved compared with the wild type, and the reaction system was successfully scaled up. When the substrate concentration was 100 g / L, the yield was close to 99% and d.e. > 99%. Compared with the reported wild-type carbonyl reductases that catalyze this substrate, the substrate concentration was increased and high diastereoselectivity was maintained. L.kefir Amit Singh, Liu Zhiqiang et al. obtained enzymes or strains with the ability to catalyze the synthesis of compound 2 from compound 1 by exploring natural carbonyl reductases or strains. Although the conversion rate and product purity were high, the substrate feeding amount was low, which was not conducive to large-scale production. The foreign enzyme preparation company Codexis used protein engineering technology to

[0006] The poor water solubility of Compound 1 and Compound 2 limits the effective contact between the substrate and the biological enzyme, and this problem has become a key bottleneck restricting the catalytic reaction efficiency. So far, adding organic solvents is a commonly used method to improve the substrate solubility in the biosynthesis process of Compound 2. However, the addition of organic solvents, especially at high doses, will have a toxic effect on the enzyme, thereby affecting the biocatalytic efficiency. The dosage of organic solvents is strictly controlled, which on the one hand limits the feeding amount of the substrate in the conversion system and affects the yield; on the other hand, due to the large precipitation of the product, the residual substrate is included, resulting in incomplete conversion of the substrate and affecting the product quality. In order to break through this bottleneck, there is an urgent need to obtain a ketoreductase with high organic solvent tolerance to realize the industrial production of the key intermediate (Compound 2) of ezetimibe. Therefore, continuing to screen ketoreductases from different biological sources or using in vitro directed evolution technology and high-throughput screening technology to evolve more efficient ketoreductases is the direction of efforts to realize the industrial production of the key intermediate of ezetimibe.

[0007] This invention is based on the patent invention with the application number 201810751489.4 and the name "An improved ketoreductase and its application" applied by our company. The original patent uses the isopropanol and ammonium formate-formate dehydrogenase system to provide coenzyme NAD + to supply H - , and further increases the raw material concentration. Even if the enzyme-catalyzed reaction time is increased or the enzyme amount is increased, there will still be more raw materials remaining. Toluene has a higher solubility for raw materials than isopropanol. Developing a more toluene-tolerant ketoreductase mutant will further increase the raw material concentration, thereby improving the production efficiency. Summary of the Invention

[0008] Object of the Invention: Aiming at the deficiencies in the enzymatic preparation of the chiral intermediate of ezetimibe in the prior art, this invention provides a ketoreductase mutant with high catalytic activity, high enantioselectivity, and high tolerance to organic solvents and substrates, as well as its application in the preparation of the chiral intermediate of ezetimibe.

[0009] On the one hand, this invention provides an improved ketoreductase, namely a ketoreductase mutant, whose tolerance to organic solvents and substrates is higher than that of the currently reported ketoreductases.

[0010] The technical solution of this invention is a ketoreductase mutant, characterized in that the amino acid sequence of the ketoreductase mutant is the amino acid sequence mutated from the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of the mutated amino acid sequence are: V at position 253 is mutated to F, and R at position 304 is mutated to I.

[0011] The amino acid sequences of the ketoreductase mutants of the present invention are: the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and EQID NO: 4.

[0012] According to one aspect of the present invention, there is provided a gene encoding any one of the above-mentioned ketoreductase mutants, and the nucleotide sequence of the gene is SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0013] According to another aspect of the present invention, there is provided a recombinant plasmid containing the nucleotide sequence of any one of the above-mentioned genes. Further, the plasmid is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32 Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C.

[0014] According to still another aspect of the present invention, there is provided a host cell containing any one of the above-mentioned recombinant plasmids, and the host cell includes prokaryotic cells, yeast, or eukaryotic cells. The prokaryotic cells are preferably Escherichia coli BL21(DE3) cells.

[0015] According to another aspect of the present invention, there is provided a method for producing (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one, which includes a reaction step of catalytic hydrogenation of 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione by a ketoreductase to form (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one, and the ketoreductase is any one of the above-mentioned ketoreductase mutants.

[0016] Advantages: By applying the technical solution of the present invention, based on the ketoreductase shown in SEQ ID NO: 1, the gene of the ketoreductase is mutated by using molecular biology methods of random mutation and site-directed saturation mutation, so as to change the amino acid sequence of the enzyme, realize the change of the enzyme structure and function, and then through the method of high-throughput directed screening, a ketoreductase with at least one of the above sites mutated is obtained. Using this ketoreductase mutant to catalyze the synthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one from the precursor ketone, in a reaction system of 150 g / L 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione, 1 g / L ketoreductase (enzyme powder), and 50% (V / V) toluene for 24 hours, the substrate conversion rate is ≥99.9%, and the product e.e. value is 100.0%. The present invention provides a technical solution with higher organic solvent tolerance and higher substrate feeding amount, which can further reduce the industrial production cost of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one and ezetimibe and improve the product quality, and has good industrial application value. Description of the Drawings

[0017] Figure 1 Chemical method for catalyzing the reduction reaction of 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione

[0018] Figure 2 Reduction reaction of 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione catalyzed by ketoreductase. Detailed Embodiments

[0019] The present invention will be further described below in conjunction with specific implementation examples, but the protection scope of the present invention is not limited thereto:

[0020] Example 1: Obtain the recombinant plasmid of the ketoreductase with the I140S mutation of strain I140S in the patent 201810751489.4 T. brockii The recombinant plasmid of the ketoreductase with the I140S mutation

[0021] Through the ketoreductase coding gene sequence of SEQ ID NO: 9 in the patent 201810751489.4, after optimizing the codons and restriction enzyme sites and commissioning a service provider to artificially synthesize the full-length gene and the restriction enzyme sites at both ends, the recombinant plasmid pET28a(+) and the full-length gene were respectively digested with restriction enzymes, the gel was recovered by cutting, ligated, and transformed into competent cells of Escherichia coli BL21(DE3), and then spread on an LB agar plate containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C. Select several single colonies into an LB medium (containing 50 mg / L kanamycin sulfate), culture overnight at 37°C, and then use a plasmid extraction kit to extract the recombinant plasmid, and perform PCR and sequencing verification to obtain the recombinant plasmid pET28a-I140S.

[0022] Example 2: Random mutation and cloning of the ketoreductase gene of the recombinant plasmid pET28a-I140S

[0023] According to the content described in Example 1, using the recombinant plasmid pET28a-I140S as a template, and designing and synthesizing primers at both ends (Table 1) according to the ketoreductase I140S coding gene using Primer 5.0. Use error-prone PCR technology (the materials and concentrations are shown in Table 2, and the reaction conditions are shown in Table 3) to obtain linear gene fragments containing a large number of base mutations. Digest these PCR products and the pET28a(+) expression plasmid with restriction enzymes respectively, recover the gel by cutting, ligate, and transform into competent cells of Escherichia coli BL21(DE3), and then spread on an LB agar plate containing 50 mg / L kanamycin sulfate and culture overnight at 37°C.

[0024] Pick the single colonies grown on the above culture dish and inoculate them into an LB liquid medium containing 50 mg / L kanamycin sulfate, shake culture overnight at 37°C, collect the bacteria for plasmid extraction, PCR identification and double digestion identification, and then name the recombinant plasmid pET28a(+)-A-Z, and perform subsequent induced expression on the Escherichia coli containing the recombinant plasmid.

[0025]

[0026]

[0027]

[0028] Example 3: Induced expression of the ketoreductase mutant and preliminary screening of enzyme activity

[0029] According to the content described in Example 2, pick monoclonal colonies on the above LB agar medium and inoculate them into a 96-well plate (pre-loaded with 0.5 mL of LB medium containing 50 mg / L kanamycin sulfate), and culture them overnight at 37°C with shaking at 220 rpm. Take 25 μL of the above culture solution and transfer it to a 48-deep well plate (pre-loaded with 2 mL of LB medium containing 50 mg / L kanamycin sulfate), culture it at 37°C with shaking at 220 rpm for 4 hours, add a certain amount of inducer isopropyl-β-D-thiogalactoside (IPTG, final concentration is 0.05 mM), induce and culture it at 28°C with shaking at 220 rpm for 14 hours, centrifuge at 5000 rpm for 20 min to collect bacterial cells, and pour out the centrifuged supernatant to obtain whole cells containing ketoreductase.

[0030] Add 100 μL of triethanolamine solution containing 1 mg / mL NAD and 100 μL of 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione solution (10 mg / mL dissolved in isopropanol) into the whole cell deep well plate respectively. Place the deep well plate at 28°C with shaking at 220 rpm for an enzymatic reaction for 26 hours. Add 900 μL of ethyl acetate, shake at 28°C with shaking at 220 rpm for 30 min to make them fully contact and mix evenly. Centrifuge at 3000 rpm for 30 min. Transfer 200 μL of the organic phase to a 96-deep well plate, add 200 μL of 2,4-dinitrophenylhydrazine solution and mix well, then place it in a constant temperature water bath at 55°C for a reaction for 15 min. After the reaction is completed, take out the deep well plate and place it in an ice-water bath to cool down to terminate the reaction. Detect the absorbance value at OD 495 value.

[0031] The mutant strain with higher enzyme activity than the parent strain in Example 3 was inoculated with 5% inoculum into 2 mL of LB medium containing 50 mg / L kanamycin sulfate, and cultured at 37°C, 220 rpm for 4 hours. 100 μL of the above culture solution was transferred to 3 mL of LB medium containing 50 mg / L kanamycin sulfate, and cultured at 37°C, 220 rpm for 2 hours. A certain amount of inducer isopropyl-β-D-thiogalactoside (IPTG, final concentration of 0.05 mM) was added, and the culture was induced at 28°C, 220 rpm for 14 hours, and the bacterial cells were collected by centrifugation at 5000 rpm for 20 min. Add 50 μL of triethanolamine solution containing 1 mg / mL NAD and 350 μL of 5-((4S)-2-oxo-4-phenyl(1, 3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1, 5-dione solution (10 mg / mL dissolved in isopropanol) to the whole cells, and place the deep-well plate at 28°C and 220 rpm for enzyme reaction for 26 hours. Add 3 mL of ethyl acetate and shake at 28°C and 220 rpm for 30 minutes to ensure full contact and mixing. Centrifuge at 3000 rpm for 30 minutes, and take the organic phase for HPLC analysis.

[0032] The mutants with better catalytic activity than I140S were selected for sequencing, the mutation sites were analyzed, and the catalytic activity was retested to determine that the catalytic activity and stereoselectivity of the mutants V253F (SEQ ID NO: 2) and R304I (SEQ ID NO: 3) were significantly improved compared with the initial I140S of this scheme. The results of the rescreening reaction are shown in Table 4. As can be seen from Table 4, the catalytic efficiency of the two ketoreductase mutants for 5-((4S)-2-oxo-4-phenyl(1, 3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1, 5-dione was 1.29 and 1.23 times that of I140S, respectively, and the product had a higher ee value.

[0033] Table 4 Comparison of the activity of ketoreductase parent and mutant enzymes in the preparation of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazacyclopentane-2-one

[0034]

[0035] Note: Table 4 * Refers to the wet weight of each ketoreductase mutant recombinant cells required to transform 1 g of substrate. 0.5 wt means 0.5 g of ketoreductase mutant recombinant wet cells are required to transform 1.0 g of substrate.

[0036] Example 5: Preparation of double mutation site mutant enzyme

[0037] Target plasmid amplification: According to the content described in Example 4, using the recombinant plasmid containing the ketoreductase-encoding gene (SEQ ID NO.7) with I140S+R304I mutations as a template, and designing and synthesizing primers to replace GTT with TTT at the mutation site according to the SEQ ID NO.7 gene sequence (Table 5), a linear gene fragment containing the TTT mutation was obtained using site-directed mutagenesis technology (materials and concentrations are shown in Table 6, reaction conditions are shown in Table 7).

[0038] Digestion of the amplification product with DpnI: Digest the amplification product with DpnI according to Table 8, place the reaction system at a constant temperature of 37 °C for 1-2 hours, and recover and purify the target amplification product by gel extraction.

[0039] Recombination reaction: After adding the materials as shown in Table 9, place the reaction at 37 °C for 30 min. After the reaction is completed, directly transform the reaction product into Escherichia coli BL21(DE3) competent cells, and then spread the transformed competent cells on an LB agar plate containing 50 mg / L kanamycin sulfate and culture overnight at 37 °C.

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Induce the expression of the mutant enzyme and verify the transformation effect as shown in Example 4. As can be seen from Table 10, the catalytic efficiency of the ketoreductase double-site mutant for 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione is 1.39 times that of the ketoreductase parent strain, and the product has a higher e.e. value.

[0046] Table 10 Comparison of the activities of the ketoreductase parent strain and the mutant enzyme in the enzymatic preparation of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

[0047]

[0048] Note: * in Table 10 refers to the wet weight of the recombinant cells of each ketoreductase mutant required to convert 1 g of substrate. 0.5 wt means that 0.5 g of wet recombinant cells of the ketoreductase mutant are required to convert 1 g of the main raw material.

[0049] Example 6: Preparation of ketoreductase mutant enzyme powder

[0050] The V253F, R304I, and V253F-R304I strains were respectively inoculated into LB medium containing 50 mg / L kanamycin sulfate at an inoculum size of 0.01% (500 mL / bottle * 10 bottles / mutant), and cultured with shaking at 37 °C and 220 rpm for 5 - 6 hours. A certain amount of inducer isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.05 mM) was added, and the culture was induced at 25 °C and 220 rpm for 16 hours. The cells were collected by centrifugation at 7000 ×g. The obtained 25 - 30 g of bacterial cells were resuspended in 100 mM phosphate buffer (pH 7.0), and the cells were disrupted using a high-pressure homogenizer. The supernatant was obtained by centrifugation at 10 °C and 10000 ×g for 20 min, and freeze-dried at -25 °C using a vacuum freeze dryer to obtain 3 - 5 g of enzyme powder, namely ketoreductase mutant enzyme powder.

[0051] Taking the V253F-R304I mutant as an example, 5.0 g of the raw material 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione and 10 - 40 mL of toluene were successively added to a 100 mL reaction flask, stirred until the raw materials were completely dissolved, 40 - 10 mL of phosphate buffer (100 mM, pH 7.0, containing 2 mM magnesium sulfate) was added, 5 mg of oxidized nicotinamide adenine dinucleotide (NAD), 4.0 g of glucose, 10 mg of glucose dehydrogenase, and 100 mg of the ketoreductase mutant enzyme powder in Example 6 were added. The pH of the system was maintained at 5.5 - 7.0 using saturated sodium carbonate, and the mixture was incubated at 30 ± 2 °C for 24 hours; 30 mL of ethyl acetate and 2 g of diatomaceous earth were added, the system was filtered by suction, the filter cake was washed with 10 mL of ethyl acetate, the filtrates were combined, allowed to stand for liquid separation, and the organic phase was concentrated to obtain the crude product of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. The detection results of the reaction conversion rate and the e.e. value of the product are shown in Table 11.

[0052] Table 11 Effects of different toluene concentrations on ketoreductase mutants

[0053]

[0054] Example 8: Application of the ketoreductase mutants shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 in the preparation of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

[0055] Add 30 g of the raw material 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione and 100 mL of toluene into a 500 mL reaction flask, stir until the raw material is completely dissolved, and add 100 mL of phosphate buffer (100 mM, pH 7.0, containing 2 mM magnesium sulfate); add 20 mg of oxidized nicotinamide adenine dinucleotide (NAD), 20 g of glucose, 0.1 g of glucose dehydrogenase and 0.2 g of the ketoreductase mutant enzyme powder in Example 6. Maintain the pH of the system at 5.5 - 7.0 using saturated sodium carbonate, and keep it warm at 30 ± 2 °C for 24 hours; add 120 mL of ethyl acetate and 9 g of diatomaceous earth, filter the system by suction, wash the filter cake with 60 mL of ethyl acetate, combine the filtrates, let it stand for liquid separation, and concentrate the organic phase to obtain the crude product of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. The detection results of the reaction conversion rate and the e.e. value of the product are shown in Table 5.

[0056] Table 12 Preparation of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one by the ketoreductase mutant enzyme method

[0057]

[0058] The results in Table 12 show that the mutant (V253F-R304I mutant (SEQ ID NO: 4)) in the enzymatic catalysis of 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione to synthesize (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one, that is, in the reaction system with 150 g / L of substrate, 50% toluene and 1 g / L of enzyme powder, after reacting for 24 hours, the conversion rate can reach over 99.9%, and the e.e. value of the product reaches 100.0%. The screened ketoreductase mutants all show extremely high stereoselectivity, organic solvent tolerance and high efficiency in the enzymatic preparation of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one.

Claims

1. An improved ketoreductase mutant, characterized in that the amino acid sequence of the ketoreductase mutant is an amino acid sequence mutated from the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of the mutated amino acid sequence are: V at position 253 is mutated to F, R at position 304 is mutated to I, or both.

2. A recombinant plasmid containing the coding gene of the ketoreductase mutant according to any one of claims 1.

3. The recombinant plasmid according to claim 2, characterized in that the plasmid is pET-28a(+), pET-28b(+), pET-28c(+), pET-5b(+), pET-15b, pET-24a(+), pET-24c(+), pET-24d(+), pET-25b(+), pET-27b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), pET-30b(+), pET-30c(+), pET-30 Xa / LIC, pET-30 EK / LIC, pET-31b(+), pET-32b(+), pET-32c(+), pET-32 EK / LIC, pET-32Xa / LIC, pET-33b(+), pET-37b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42b(+), pET-42c(+), pET-43.1a(+), pET-43.1b(+), pET-43.1c(+), pET-43.1 EK / LIC, pET-44a(+), pET-44b(+), pET-44c(+), pET-44 EK / LIC, pET-45b(+), pET-46 EK / LIC, pET-47b(+), pET-48b(+), pET-49b(+), pET-51b(+), pET-52b(+), pQE30, pQE31, pQE32, pQE40, pBV220, pBV221, pCold-GST, pCold IV, pCold-GST or pTrcHis C.

4. A host cell containing the recombinant plasmid according to claim 3.

5. The host cell according to claim 4, characterized in that the host cell includes a prokaryotic cell or a eukaryotic cell, and the prokaryotic cell is Escherichia coli BL21(DE3) cell.

6. Use of the ketoreductase mutant according to claim 1 in reducing the substrate 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one.

7. The use according to claim 6, characterized in that the substrate, toluene, phosphate buffer, coenzyme, hydrogen donor, and ketoreductase mutant are added during the reduction process.

8. The use according to claim 7, characterized in that the coenzyme is oxidized nicotinamide adenine dinucleotide (NAD), and the hydrogen donor is glucose and glucose dehydrogenase.

9. The use according to any one of claims 6 or 7, characterized in that the pH of the reduction reaction system is maintained at 5.5 to 7.0, and the volume of toluene in the reduction system does not exceed 50%.

Images