Ketoreductase mutant and application thereof in preparation of (R)-3-iodocyclopent-2-ene-1-ol

Abstract

The invention discloses a ketoreductase mutant and application of the ketoreductase mutant in preparation of (R)-3-iodocyclopent-2-ene-1-ol. Compared with an amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the ketoreductase mutant has substitution mutation at one or more key positions of the 49th position, the 95th position, the 98th position, the 101th position, the 110th position, the 129th position, the 167th position, the 168th position and the 169th position. The invention also provides a method for preparing (R)-3-iodocyclopent-2-ene-1-ol by using the ketoreductase mutant disclosed by the invention, and the method for preparing (R)-3-iodocyclopent-2-ene-1-ol by using the ketoreductase mutant disclosed by the invention as a catalyst through asymmetric reduction of 3-iodo-2-cyclopentenone has the advantages that the substrate conversion rate reaches 98.8%, the yield is up to 98.8%, and the yield is up to 98.8%. The ee value of the reduction product is greater than 98%, the reaction condition is mild, the method is environment-friendly, and the method has a better industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a ketoreductase mutant and application thereof in the preparation of (R)-3-iodocyclopent-2-ene-1-ol. Background Art

[0002] Ketoreductase (KRED), also known as carbonyl reductase, is an enzyme that catalyzes the reversible redox reaction between aldehydes / ketones and alcohols, and requires coenzymes such as NAD (H) or NADP (H) as hydrogen transfer agents. Structurally, it has an (α / β) 8-barreled structure and an active site Asp-Tyr-Lys-His. Most of the expression is soluble, and it is widely present in different animals, plants, yeasts and bacteria in nature.

[0003] Ketoreductases are mainly divided into three categories, namely short-chain dehydrogenase / reductases (SDRs), medium-chain dehydrogenases / reductases (MDRs) and aldo-ketoreductases (AKRs); the catalytic functions of the three are similar, but they differ greatly in structure and properties.

[0004] The use of ketoreductase for biotransformation has the advantages of strong substrate specificity, strong chiral selectivity, high conversion efficiency, low generation of by-products, mild reaction conditions and low environmental pollution. With the rapid development of biotechnology, more and more excellent KRED recombinant enzymes have been discovered and applied to the research of chiral alcohols, pharmaceutical and chemical intermediates.

[0005] (R)-3-iodocyclopent-2-ene-1-ol is a synthetic intermediate of a compound with medical value. At present, (R)-3-iodocyclopent-2-ene-1-ol is mainly prepared by chemical methods, and the chemical synthesis method requires operations such as layering, filtering, drying and fractionation, and the process is complicated and the steps are cumbersome. From the perspective of economy and environmental protection, it is not suitable for industrial large-scale production.

[0006] Therefore, there is an urgent need in the art for a method for preparing (R)-3-iodocyclopent-2-ene-1-ol that is suitable for large-scale industrial production, has high safety, and is simple to operate. Summary of the invention

[0007] Compared with traditional chemical synthesis methods, bioenzyme catalysis has the advantages of high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, and environmental friendliness. Therefore, the present invention provides a ketoreductase mutant and its application in the preparation of (R)-3-iodocyclopent-2-ene-1-ol, which improves substrate conversion rate and yield, and has the characteristics of simple operation, safety and environmental protection.

[0008] The present invention provides a ketoreductase mutant, wherein the ketoreductase mutant has any one of the amino acid sequences shown in (I) or (II):

[0009] (I) an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:1;

[0010] (II) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to amino acids 49 to 169 of the amino acid sequence shown in SEQ ID NO: 1;

[0011] Wherein, the ketoreductase mutant has ketoreductase activity.

[0012] Specifically, the substitution is the substitution of 1-9 amino acids.

[0013] Specifically, the substitution is a substitution mutation at any one or at least two of the amino acid positions 49, 95, 98, 101, 110, 129, 167, 168 or 169.

[0014] Preferably, the substitution is a mutation at one or more key positions of position 49, position 95, position 98, position 101, position 110, position 129, position 167, position 168, position 169. Preferably, the substitution is a conservative substitution. In some embodiments, the wild-type P at position 49 is substituted with a non-polar amino acid, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably substituted with isoleucine, phenylalanine, tryptophan, proline; more preferably substituted with isoleucine. The wild-type F at position 95 is substituted with an uncharged polar amino acid, serine, threonine, tyrosine, asparagine, glutamine, cysteine ​​and glycine, preferably substituted with asparagine, tyrosine, cysteine, more preferably substituted with tyrosine. The wild-type P at position 98 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, and proline, preferably substituted with isoleucine, phenylalanine, tryptophan, and proline; more preferably substituted with isoleucine. The wild-type H at position 101 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, and methionine, preferably substituted with methionine, isoleucine, phenylalanine, tryptophan, and proline; more preferably substituted with methionine. The wild-type L at position 110 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and proline, preferably substituted with an aromatic non-polar amino acid, such as phenylalanine and tryptophan; more preferably substituted with phenylalanine. The wild-type S at position 129 is substituted with an aliphatic non-polar amino acid, such as alanine, leucine, valine, proline or isoleucine, preferably substituted with isoleucine, leucine, valine; more preferably substituted with valine. The wild-type F at position 167 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably substituted with leucine, phenylalanine, tryptophan, proline; more preferably substituted with leucine. The wild-type M at position 168 is substituted with other non-polar amino acids, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably substituted with alanine, valine, leucine, isoleucine; more preferably substituted with alanine. The wild-type R at position 169 is substituted with an uncharged polar amino acid, such as serine, threonine, tyrosine, asparagine, glutamine, cysteine ​​and glycine, preferably substituted with asparagine, glutamine, cysteine; more preferably substituted with glutamine.

[0015] Specifically, the substitution mutation is selected from one or more of the following substitution mutations: P49I, F95Y, F95M, T98I, H101M, L110Y, S129L, F167L, M168A, M168S, M168V and R169Q.

[0016] Specifically, the amino acid sequence of the ketoreductase mutant is shown in SEQ ID NO:2 and SEQ ID NO:3.

[0017] The second aspect of the present invention provides a nucleic acid molecule, the nucleotide sequence of the nucleic acid molecule comprising:

[0018] (1) a nucleotide sequence encoding the ketoreductase mutant described in the first aspect;

[0019] (2) A nucleotide sequence complementary to the nucleotide sequence described in (1).

[0020] Specifically, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:6.

[0021] The third aspect of the present invention provides a nucleic acid construct, which contains the nucleic acid molecule described in the second aspect, and the nucleic acid construct is an expression cassette. In addition to the nucleic acid molecule, the expression cassette may also contain a transcription termination sequence and a promoter. The promoter may be any of various promoters known in the art, as long as it is suitable for expressing the ketoreductase of the present invention in the desired host. Those skilled in the art can select an appropriate promoter according to the host cell used to construct the expression cassette and recombinant vector of the present invention.

[0022] The fourth aspect of the present invention provides a recombinant vector, the recombinant vector contains at least one copy of the nucleic acid molecule as described in the second aspect, and the recombinant vector is a recombinant cloning vector or a recombinant expression vector. The recombinant vector may contain other regulatory elements, including but not limited to enhancers, multiple cloning sites, transcription terminators, resistance genes, etc. The corresponding vector skeleton with the required regulatory elements can be selected according to different purposes, and the nucleic acid molecule or nucleic acid construct of the present invention is cloned into the skeleton, thereby constructing the recombinant vector of the present invention.

[0023] The ketoreductase described herein can be prepared by preparing nucleic acid molecules, constructing nucleic acid constructs and recombinant vectors using methods well known in the art, and expressing using conventional methods.

[0024] The fifth aspect of the present invention provides a host cell, which contains the recombinant vector described in the fourth aspect and / or expresses the ketoreductase mutant described in the first aspect, and the host cell is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0025] The sixth aspect of the present invention provides an enzyme preparation, wherein the enzyme preparation contains the ketoreductase mutant described in the first aspect.

[0026] In a seventh aspect of the present invention, a method for preparing (R)-3-iodocyclopent-2-ene-1-ol is provided, wherein the ketoreductase is a ketoreductase derived from Pseudomonas aeruginosa or a ketoreductase mutant as described in the first aspect. The method for preparing (R)-3-iodocyclopent-2-ene-1-ol comprises the following steps:

[0027] In the presence of a cosolvent, glucose and a coenzyme, using the ketoreductase as a catalyst, asymmetrically reducing the compound of formula (i) to obtain a compound of formula (ii) or formula (iii);

[0028] Wherein, the compound of formula (i) is The compound of formula (ii) is The compound of formula (iii) is

[0029] Specifically, the amino acid sequence of the ketoreductase from Pseudomonas aeruginosa is shown in SEQ ID NO:1.

[0030] Specifically, the ketoreductase includes a ketoreductase having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the NCBI accession number is ABB91667.

[0031] Specifically, the cosolvent is selected from a combination of one or more of dimethyl sulfoxide, alcohol solvents and toluene, the reaction system contains metal ions and uses a phosphate buffer or a Tris-HCl buffer. The cosolvent used should be miscible with water to further increase the solubility of the substrate.

[0032] Preferably, the co-solvent is dimethyl sulfoxide.

[0033] Preferably, the metal ion is zinc ion, manganese ion or cobalt ion; more preferably, the metal ion is manganese ion.

[0034] Preferably, the concentration of the phosphate buffer is 10-500 mM; more preferably, the concentration of the phosphate buffer is 50-200 mM.

[0035] Specifically, the reaction conditions of the reaction are: the pH of the reaction system is 6 to 10, the reaction temperature is 10° C. to 50° C., and the reaction time is 0.1 to 96 hours.

[0036] Preferably, the reaction conditions are: the pH of the reaction system is 6.5 to 7.5, the reaction temperature is 20° C. to 45° C., more preferably 25° C. to 32° C., and the reaction time is 12 to 25 hours.

[0037] Specifically, the amount of the catalyst is 0.01-10 times the weight of the compound of formula (i), the amount of the metal ion is 0.01-2% the weight of the compound of formula (i), the amount of the coenzyme is 0.01-0.5 times the weight of the compound of formula (i), and the amount of glucose is 0.01-10 times the weight of the compound of formula (i).

[0038] Preferably, the amount of the catalyst is 1-4 times the weight of the compound of formula (i), the amount of the metal ion is 0.05-1% of the weight of the compound of formula (i), the amount of the coenzyme is 0.05-0.1 times the weight of the compound of formula (i), and the amount of glucose is 1-6 times the weight of the compound of formula (i).

[0039] Preferably, the coenzymes are glucose dehydrogenase and NADP.

[0040] The eighth aspect of the present invention provides a ketoreductase or an enzyme preparation thereof derived from Pseudomonas aeruginosa and the use of the ketoreductase mutant described in the first aspect in the preparation of chiral drugs or intermediates thereof.

[0041] The main advantages of the present invention include:

[0042] 1) The ketoreductase mutant disclosed in the present application is used as a catalyst for the asymmetric reduction of 3-iodo-2-cyclopentenone by a bioenzymatic method to prepare (R)-3-iodocyclopent-2-ene-1-ol, with a substrate conversion rate of 98.8% and an ee value of the reduction product greater than 98%.

[0043] 2) The method for preparing (R)-3-iodocyclopent-2-ene-1-ol disclosed in the present application adopts a biological enzyme method to asymmetric reduce 3-iodo-2-cyclopentenone, which reduces the reaction cost. Under the reaction conditions provided in the present application, the reaction steps are short, the reaction conditions are mild and the operation is simple, the safety is high, the pollution to the environment is small, and it is easy to industrialize production. Therefore, the present method has excellent industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The SFC spectrum of (R)-3-iodocyclopent-2-ene-1-ol and (S)-3-iodocyclopent-2-ene-1-ol after the reaction according to the method described in Example 1. The peak at t=1.473min is (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.581min is (S)-3-iodocyclopent-2-ene-1-ol.

[0045] Figure 2This is the SFC spectrum of (R)-3-iodocyclopent-2-ene-1-ol and (S)-3-iodocyclopent-2-ene-1-ol after the reaction according to the method described in Example 2. The peak at t=1.449min is (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.548min is (S)-3-iodocyclopent-2-ene-1-ol.

[0046] Figure 3 The SFC spectrum of (R)-3-iodocyclopent-2-ene-1-ol and (S)-3-iodocyclopent-2-ene-1-ol after the reaction according to the method described in Example 3. The peak at t=1.490min is (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.593min is (S)-3-iodocyclopent-2-ene-1-ol.

[0047] Figure 4 The SFC spectrum of (R)-3-iodocyclopent-2-ene-1-ol and (S)-3-iodocyclopent-2-ene-1-ol after the reaction according to the method described in Example 4. The peak at t=1.485min is (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.587min is (S)-3-iodocyclopent-2-ene-1-ol.

[0048] Figure 5 This is the SFC spectrum of (R)-3-iodocyclopent-2-ene-1-ol and (S)-3-iodocyclopent-2-ene-1-ol after the reaction according to the method described in Example 5. The peak at t=1.460min is (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.561min is (S)-3-iodocyclopent-2-ene-1-ol.

[0049] Figure 6 This is the SFC spectrum of (S)-3-iodocyclopent-2-en-1-ol after the reaction according to the method described in Example 6. The peak at t=1.563 min is (S)-3-iodocyclopent-2-en-1-ol. DETAILED DESCRIPTION

[0050] The present invention discloses a ketoreductase mutant, and it is found that the ketoreductase mutant can be used to prepare the compound shown in the following formula ii or iii with a higher conversion rate and chirality:

[0051]

[0052] In the present invention, the ketoreductase is from Starmerella magnoliae, and exemplary ketoreductases include ketoreductases having an amino acid sequence as shown in SEQ ID NO: 1, and also include mutants of the ketoreductases of the amino acid sequence as shown in SEQ ID NO: 1, which have an amino acid sequence with at least 75% identity, or preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity compared to the amino acid sequence as shown in SEQ ID NO: 1. In the art, sequence identity can be calculated using software commonly used in the art, such as BLAST (from NCBI) with software default parameters.

[0053] Furthermore, the ketoreductase mutant of the present invention is a mutant derived from SEQ ID NO: 1 obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to multiple amino acid mutations and retaining the ketoreductase activity possessed by SEQ ID NO: 1. The one or more amino acid mutations include amino acid mutations within 20, preferably within 15, more preferably within 10, more preferably within 8, more preferably within 5, and more preferably within 3, such as substitution, insertion or deletion of amino acid residues. Among them, the preferred mutation is substitution.

[0054] In the present invention, exemplary ketoreductase mutants include, but are not limited to, the ketoreductase with accession number ABB91667.

[0055] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Unless otherwise specified, the raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0056] The present invention is further described below in conjunction with the preparation examples and multiple examples. It should be understood that these examples are merely illustrative and are not intended to limit the present invention. The methods, materials and reagents used in the examples are, unless otherwise stated, conventional methods in the art, and materials and reagents available from commercial sources.

[0057] Preparation Example

[0058] The amino acid sequence shown in SEQ ID NO:1 (the coding sequence is shown in SEQ ID NO:4) was used as the parent, and the directed evolution transformation was carried out by using roller PCR, iterative saturation mutation, and combined mutation strategies. Then the mutant was transformed into Escherichia coli BL21 (DE3) competent cells, and evenly coated on LB agar plates containing 50ug / ml kanamycin, and placed in a 37°C incubator for static culture for 18h. The mutants on the transformed plate were picked into a 96-well plate with a sterile toothpick and cultured overnight in a shaker at 37°C and 250rpm. 50 microliters of bacterial solution were drawn from the holes of the primary plate and inoculated into the corresponding holes of the secondary plate. After culturing at 37°C and 250rpm for 2-3h, IPTG with a final concentration of 0.2mM was added, and the mutants were cultured at 30°C for 20h to obtain the corresponding mutants for high-throughput screening. Combined with HPLC and GC detection rescreening, mutants with significantly improved activity and stability were identified for gene sequencing. The sequencing results are shown in SEQ ID NO:2 and SEQ ID NO:3, and the coding sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0059] The relevant sequence information involved in this application is as follows:

[0060] SEQ ID NO: 1, wild-type ketoreductase

[0061] MTTTSNALVTGGSRGIGAASAIKLAQEGYSVTLASRSVDKLNEVKAKLPIVQDGQKHYIWELDLADVEAASSFKGAPLPASSYDVFVSNAGVAAFSPTADHDDKEWQNLLAVNLSSPIAL TKALLKDVSERPADNPLQIIYISSVAGLHGAAQVAVYSASKAGLDGFMRSVAREVGPKGIHVNSINPGTTKTEMTAGIEALPDLPIKGWIEPEAIADAVLFLAKSKNITGTNIVVDNGLIA

[0062] SEQ ID NO: 2, Ketoreductase mutant 1

[0063] MTTTSNALVTGGSRGIGAASAIKLAQEGYSVTLASRSVDKLNEVKAKLIIVQDGQKHYIWELDLADVEAASSFKGAPLPASSYDVFVSNAGVAAFSPTADMDDKEWQNLLAVNLSSPIALTKALLKDVSERPADNPLQIIYISSVAGLHGAAQVAVYSASKAGLDGFARSVAREVGPKGIHVNSINPGTTKTEMTAGIEALPDLPIKGWIEPEAIADAVLFLAKSKNITGTNIVVDNGLIA

[0064] SEQ ID NO:3, Ketoreductase Mutant 2

[0065] MTTTSNALVTGGSRGIGAASAIKLAQEGYSVTLASRSVDKLNEVKAKLPIVQDGQKHYIWELDLADVEAASSFKGAPLPASSYDVFVSNAGVAAFSPIADHDDKEWQNLLAVNLSSPIALTKALLKDVLERPADNPLQIIYISSVAGLHGAAQVAVYSASKAGLDGFSRSVAREVGPKGIHVNSINPGTTKTEMTAGIEALPDLPIKGWIEPEAIADAVLFLAKSKNITGTNIVVDNGLIA

[0066] SEQ ID NO:4, Coding DNA Sequence of Wild-Type Ketoreductase

[0067] ATGACTACCACCTCTAACGCACTTGTAACCGGCGGTTCACGTGGTATCGGTGCGGCTTCTGCCATTAAACTGGCGCAGGAGGGCTATTCCGTTACGCTGGCGTCCCGTTCTGTTGATAAACTGAACGAAGTTAAAGCCAAACTGCCCATCGTACAGGATGGCCAAAAACACTACA TTTGGGAACTGGACCTTGCCGATGTTGAAGCTGCCAGCAGCTTTAAAGGTGCTCCGCTGCCGGCAAGCTCTTACGATGTGTTCGTATCCAACGCGGGCGTTGCCGCCTTTTCCCCGACCGCGGACCACGACGACAAAGAGTGGCAGAACCTGCTGGCAGTAAACCTGAGTTCACCGATCGCTCTCACCAAGGCCCTGCTGAAAGATGTGAGCGAACGTCCGGCTGATAATCCGCTTCAAATTATCTACATCTCCTCTGTTGCCGGCCTGCACGGCGCGGCCCAGGTTGCTGTTTACTCCGCGTCCAAGGCCGGCCTGGATGGGTTCATGCGCTCTGTTGCACGCGAAGTAGGTCCGAAAGGCATCCATGTAAACAGCATCAACCCTGGTACGACGAAGACGGAAATGACCGCAGGCATCGAAGCACTGCCGGATCTGCCGATCAAAGGCTGGATTGAACCGGAAGCGATCGCCGATGCCGTGCTGTTTCTGGCCAAATCTAAAAATATCACGGGTACCAACATCGTGGTTGACAACGGCCTGATCGCGTGA

[0068] SEQ ID NO:5, Coding DNA sequence of ketoreductase mutant 1

[0069] ATGACTACCACCTCTAACGCACTTGTAACCGGCGGTTCACGTGGTATCGGTGCGGCTTCTGCCATTAAACTGGCGCAGGAGGGCTATTCCGTTACGCTGGCGTCCCGTTCTGTTGATAAACTGAACGAAGTTAAAGCCAAACTGCCCGTAGTACAGGATGGCCAAAAACACTACATTTGGGAACTGGACCTTGCCGATGTTGAAGCTGCCAGCAGCTTTAAAGGTGCTCCGCTGCCGGCAAGCTCTTACGATGTGTTCGTATCCAACGCGGGCGTTGCCGCCTTTTCCCCGACCGCGGACATGGACGACAAAGAGTGGCAGAACCTGCTGGCAGTAAACCTGAGTTCACCGATCGCTCTCACCAAGGCCCTGCTGAAAGATGTGAGCGAACGTCCGGCTGATAATCCGCTTCAAATTATCTACATCTCCTCTGTTGCCGGCCTGCACGGCGCGGCCCAGGTTGCTGTTTACTCCGCGTCCAAGGCCGGCCTGGATGGGTTCGCGCGCTCTGTTGCACGCGAAGTAGGTCCGAAAGGCATCCATGTAAACAGCATCAACCCTGGTACGACGAAGACGGAAATGACCGCAGGCATCGAAGCACTGCCGGATCTGCCGATCAAAGGCTGGATTGAACCGGAAGCGATCGCCGATGCCGTGCTGTTTCTGGCCAAATCTAAAAATATCACGGGTACCAACATCGTGGTTGACAACGGCCTGATCGCGTGA

[0070] SEQ ID NO:6, Coding DNA sequence of ketoreductase mutant 2

[0071] ATGACTACCACCTCTAACGCACTTGTAACCGGCGGTTCACGTGGTATCGGTGCGGCTTCTGCCATTAAACTGGCGCAGGAGGGCTATTCCGTTACGCTGGCGTCCCGTTCTGTTGATAAACTGAACGAAGTTAAAGCCAAACTGCCCATCGTACAGGATGGCCAAAAACACTACA TTTGGGAACTGGACCTTGCCGATGTTGAAGCTGCCAGCAGCTTTAAAGGTGCTCCGCTGCCGGCAAGCTCTTACGATGTGTTCGTATCCAACGCGGGCGTTGCCGCCTTTTCCCCGATCGCGGACCACGACGACAAA GAGTGGCAGAACCTGCTGGCAGTAAAACCTGAGTTCACCGATCGCTCTCACCAAGGCCCTGCTGAAAGATGTGCTCGAACGTCCGGCTGATAATCCGCTTCAAATTATCTACATCTCCTCTGTTGCCGGCCTGCACGGC GCGGCCCAGGTTGCTGTTTACTCCGCGTCCAAGGCCGGCCTGGATGGGTTCTCGCGCTCTGTTGCACGCGAAGTAGGTCCGAAAGGCATCCATGTAAACAGCATCAACCCTGGTACGACGAAGACGGAAATGACCGCA GGCATCGAAGCACTGCCGGATCTGCCGATCAAAGGCTGGATTGAACCGGAAGCGATCGCCGATGCCGTGCTGTTTCTGGCCAAATCTAAAAATATCACGGGTACCAACATCGTGGTTGACAACGGCCTGATCGCGTGA

[0072] Example 1

[0073] Prepare 200 mM phosphate buffer solution: weigh 27.84 g potassium phosphate trihydrate and 10.62 g potassium dihydrogen phosphate into a 1 L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1 L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0074] 50 mg of glucose, 2 mg of NADP, 4 mg of glucose dehydrogenase, and 10 mg of ketoreductase powder (amino acid sequence as shown in SEQ ID NO: 1) were weighed in sequence and added into a 4 mL glass tube containing 2 mL of 100 mM phosphate buffer solution, and vortexed to mix.

[0075] Weigh 20 mg of substrate (3-iodo-2-cyclopentenone) and add it to 80 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0076] The glass tube was tightly capped and placed in a 20°C 800rpm shaker. It was taken out after 20 hours. 100uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of methanol was added, vortexed, mixed, and then centrifuged at 12000rpm for 2 minutes. 300uL of the supernatant was taken for SFC detection. The peak at t=1.473min was (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.581min was (S)-3-iodocyclopent-2-ene-1-ol. The ee value was 4.98%, and the conversion rate was 14.86%.

[0077] Example 2

[0078] Prepare 200 mM phosphate buffer solution: weigh 27.84 g potassium phosphate trihydrate and 10.62 g potassium dihydrogen phosphate into a 1 L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1 L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0079] 30 mg of glucose, 4 mg of NADP, 2 mg of glucose dehydrogenase, and 1 mg of ketoreductase powder (amino acid sequence as shown in SEQ ID NO: 2) were weighed in sequence and added into a 4 mL glass tube containing 2 mL of 200 mM phosphate buffer solution, and vortexed to mix.

[0080] Weigh 20 mg of substrate (3-iodo-2-cyclopentenone) and add it to 10 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0081] The glass tube was tightly capped and placed in a 25°C 1000rpm shaker, and taken out after 20 hours. 100uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of methanol was added, vortexed, mixed, and then centrifuged at 12000rpm for 2min. 300uL of the supernatant was taken for SFC detection. The peak at t=1.449min was (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.548min was (S)-3-iodocyclopent-2-ene-1-ol. The ee value was 70.35%, and the conversion rate was 84.14%.

[0082] Example 3

[0083] Prepare 200 mM phosphate buffer solution: weigh 27.84 g potassium phosphate trihydrate and 10.62 g potassium dihydrogen phosphate into a 1 L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1 L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0084] 20 mg of glucose, 0.5 mg of NADP, 1 mg of glucose dehydrogenase, and 1.2 mg of ketoreductase powder (amino acid sequence as shown in SEQ ID NO: 3) were weighed in sequence and added into a 4 mL glass tube containing 2 mL of 200 mM phosphate buffer solution, and vortexed to mix.

[0085] Weigh 40 mg of substrate (3-iodo-2-cyclopentenone) and add it to 30 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0086] The glass tube was tightly capped and placed in a 32°C 1000rpm shaker, and taken out after 20 hours. 100uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of methanol was added, vortexed, mixed, and then centrifuged at 12000rpm for 2min. 300uL of the supernatant was taken for SFC detection. The peak at t=1.490min was (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.593min was (S)-3-iodocyclopent-2-ene-1-ol. The ee value was 97.33%, and the conversion rate was 71.37%.

[0087] Example 4

[0088] Prepare 200 mM phosphate buffer solution: weigh 27.84 g potassium phosphate trihydrate and 10.62 g potassium dihydrogen phosphate into a 1 L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1 L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0089] 40 mg of glucose, 0.5 mg of NADP, 1 mg of glucose dehydrogenase, 0.1 mg of magnesium chloride, and 2 mg of ketoreductase powder (amino acid sequence as shown in SEQ ID NO: 3) were weighed in sequence and added into a 4 mL glass tube containing 2 mL of 50 mM phosphate buffer solution, and vortexed to mix.

[0090] Weigh 40 mg of substrate (3-iodo-2-cyclopentenone) and add it to 40 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0091] The glass tube was tightly capped and placed in a 35°C 1000rpm shaker, and taken out after 20 hours. 100uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of methanol was added, vortexed, mixed, and then centrifuged at 12000rpm for 2min. 300uL of the supernatant was taken for SFC detection. The peak at t=1.485min was (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.587min was (S)-3-iodocyclopent-2-ene-1-ol. The ee value was 97.96%, and the conversion rate was 98.8%.

[0092] Example 5

[0093] Take about 20 mg of the bacterial cells (after induction expression, containing the ketoreductase shown in the amino acid sequence SEQ ID NO: 3), treat the cells with 270 Ul of phosphate buffer solution (containing 1 mg / mL nuclease and 2 mg / mL lysozyme), and place them in a shaker at 30°C and 1000 rpm for 2 hours. Take them out and centrifuge them at 4000 rpm for 15 minutes, take 80 uL of the supernatant and add it to a new 4 mL glass tube.

[0094] Weigh 4 mg of substrate (3-iodo-2-cyclopentenone) and add it to 4 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0095] The glass tube was tightly capped and placed in a shaking table at 30°C and 1000 rpm, and taken out after 20 hours. 200uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of acetonitrile was added, vortexed, mixed, and then centrifuged at 12000rpm for 2 minutes. 300uL of the supernatant was taken for SFC detection. The peak at t=1.460min was (R)-3-iodocyclopent-2-ene-1-ol, and the peak at t=1.561min was (S)-3-iodocyclopent-2-ene-1-ol. The ee value was 96.61%, and the conversion rate was 44.53%.

[0096] Example 6

[0097] Prepare 200 mM phosphate buffer solution: weigh 27.84 g potassium phosphate trihydrate and 10.62 g potassium dihydrogen phosphate into a 1 L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1 L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0098] 10 mg of glucose, 3.2 mg of NADP, 1 mg of glucose dehydrogenase, and 13 mg of ketoreductase powder (amino acid sequence as shown in SEQ ID NO: 3) were weighed in sequence and added into a 4 mL glass tube containing 2 mL of 200 mM phosphate buffer solution, and vortexed to mix.

[0099] Weigh 40 mg of substrate (3-iodo-2-cyclopentenone) and add it to 40 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0100] The glass tube was capped tightly and placed in a 37°C 1000rpm shaker. After 20 hours, it was taken out. 20uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of methanol was added, vortexed, mixed, and then centrifuged at 12000rpm for 2 minutes. 300uL of the supernatant was taken for SFC detection. The peak at t=1.563min was (S)-3-iodocyclopent-2-ene-1-ol, with an ee value of -100% and a conversion rate of 98.54%.

[0101] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A ketoreductase mutant, characterized in that: The ketoreductase mutant has any one of the amino acid sequences shown in (I) or (II): (I) an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO:1; (II) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to amino acids 49 to 169 of the amino acid sequence shown in SEQ ID NO: 1; Wherein, the ketoreductase mutant has ketoreductase activity.

2. The ketoreductase mutant according to claim 1, characterized in that The substitution is for 1 to 9 amino acids.

3. The ketoreductase mutant according to claim 1, characterized in that The substitution is a substitution mutation at any one or at least two of the amino acid positions among position 49, position 95, position 98, position 101, position 110, position 129, position 167, position 168 or position 169.

4. The ketoreductase mutant according to claim 3, characterized in that The substitution mutation is selected from one or more of the following substitution mutations: P49I, F95Y, F95M, T98I, H101M, L110Y, S129L, F167L, M168A, M168S, M168V and R169Q.

5. The ketoreductase mutant according to claim 4, characterized in that The amino acid sequences of the ketoreductase mutants are shown in SEQ ID NO:2 and SEQ ID NO:

3.

6. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid molecule comprises: (1) a nucleotide sequence encoding the ketoreductase mutant according to any one of claims 1 to 5; (2) A nucleotide sequence complementary to the nucleotide sequence described in (1).

7. The nucleic acid molecule according to claim 6, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

6.

8. A nucleic acid construct, characterized in that The nucleic acid construct contains the nucleic acid molecule according to claim 6, and the nucleic acid construct is an expression cassette.

9. A recombinant vector, characterized in that: The recombinant vector contains at least one copy of the nucleic acid molecule according to claim 6, and the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

10. A host cell, characterized in that The host cell contains the recombinant vector according to claim 9, and / or expresses the ketoreductase mutant according to any one of claims 1 to 5, and the host cell is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells.

11. An enzyme preparation, characterized in that The enzyme preparation contains the ketoreductase mutant according to any one of claims 1 to 5.

12. Use of a ketoreductase in the preparation of (R)-3-iodocyclopent-2-en-1-ol, characterized in that: The ketoreductase is a ketoreductase derived from Magnolia grandiflora or a ketoreductase mutant according to any one of claims 1 to 5. The method for preparing (R)-3-iodocyclopent-2-en-1-ol comprises the following steps: In the presence of a cosolvent, glucose and a coenzyme, using the ketoreductase as a catalyst, asymmetrically reducing the compound of formula (i) to obtain a compound of formula (ii) or formula (iii); Wherein, the compound of formula (i) is The compound of formula (ii) is The compound of formula (iii) is 13. The use according to claim 12, characterized in that: The co-solvent is selected from a combination of one or more of dimethyl sulfoxide, alcohol solvents and toluene. The reaction system contains metal ions and uses a phosphate buffer.

14. The use according to claim 12, characterized in that: The reaction conditions of the reaction are as follows: the pH of the reaction system is 6 to 10, the reaction temperature is 10° C. to 50° C., and the reaction time is 0.1 to 96 hours.

15. The use according to claim 13, characterized in that: The amount of the catalyst is 0.01-10 times the weight of the compound of formula (i), the amount of the metal ion is 0.01-2% the weight of the compound of formula (i), the amount of the coenzyme is 0.01-0.5 times the weight of the compound of formula (i), and the amount of glucose is 0.01-10 times the weight of the compound of formula (i).

16. Use of the ketoreductase derived from Pseudomonas aeruginosa or an enzyme preparation thereof and the ketoreductase mutant according to any one of claims 1 to 5 in the preparation of chiral drugs or intermediates thereof.

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