Alkene reductase mutant and use thereof in catalytic preparation of intermediate for synthesis of brivaracetam

Abstract

The present invention belongs to the technical field of biocatalysts. Disclosed are an alkene reductase mutant and the use thereof in the catalytic preparation of an intermediate for the synthesis of brivaracetam. The amino acid sequence of the alkene reductase mutant of the present invention is as shown in SEQ ID NO. 1. A compound enzyme composed of the mutant, carbonyl reductase K1 and glucose dehydrogenase can, with the compound 5-hydroxy-4-n-propyl-2-furanone as a substrate, reduce double bonds and remove hydroxyl, so as to obtain the compound (R)-4-propyl dihydrofuran-2(3H)-one; and the ee value of the (R)-4-propyl dihydrofuran-2(3H)-one obtained by means of catalysis reaches 93.0%, and (R)-4-propyl dihydrofuran-2(3H)-one having an ee value of greater than 99.8% can be further obtained by means of the crystallization of diastereoisomers.

Description

olefin reductase mutants and their applications in the catalytic preparation of buvasidan synthetic intermediates Technical Field

[0001] This invention belongs to the field of biocatalyst technology, and specifically relates to an olefin reductase mutant and its use in the catalytic preparation of buvasidan synthetic intermediates. Background Technology

[0002] Brivaceratam, also known as brivaracetam, chemically named (S)-2-((R)-2-oxo-4-propylpyrrolidine-1-yl)butyramide, is a structural analog of levetiracetam. Developed by UC Pharma in Belgium, brivaracetam is a third-generation antiepileptic drug that selectively binds to central synaptic vesicle protein 2A, regulating the release of neurotransmitters at the synapse to exert its antiepileptic effect. It boasts advantages such as high bioavailability and good safety profile.

[0003] (R)-4-n-propyldihydrofuran-2-one is a key intermediate in the synthesis of buvascarb. Due to its simple structure and the small steric hindrance difference between the five-membered ring lactone intermediate and its enantiomer, its preparation and separation are both challenging. Therefore, developing a novel asymmetric catalytic synthesis method to obtain buvascarb intermediates with high optical purity remains an urgent problem to be solved.

[0004] Patent WO2016191435A1 introduces a chiral center using (R)-epoxychloropropane as the starting material, and obtains (R)-4-propyldihydrofuran-2(3H)-one via nucleophilic substitution, Grignard reaction, and decarboxylation. This route uses inexpensive and readily available raw materials, but the Grignard reaction requires stringent conditions, necessitating multiple vacuum distillations throughout the process, and the product's ee (efficiency) is only 98%, which still needs further improvement.

[0005] Patent CN105801530 uses the chiral raw material (R)-2-aminovaleric acid, which is substituted with hydrogen bromide to obtain (R)-2-bromovaleric acid. Then, the carboxyl group is reduced with diborane, the hydroxyl group is protected with tert-butyldimethylsilyl, followed by nucleophilic substitution, deprotection, hydrolysis, and esterification to form a ring, yielding (R)-4-propyldihydrofuran-2(3H)-one. This route uses highly toxic hydrogen bromide and diborane, posing safety hazards.

[0006] Patent CN106008411A uses (S)-4-benzyl-2-oxazolidinone as a chiral auxiliary agent, reacting it with valeric acid and tert-valeryl chloride, followed by reaction with tert-butyl bromoacetate under strong base conditions. The oxazolidinone cofactor is then removed via hydrogen peroxide and lithium hydroxide. Finally, the carboxyl group is reduced, and cyclization yields (R)-4-propyldihydrofuran-2(3H)-one. This route requires ultra-low temperatures of -70°C, making the reaction conditions quite harsh. The use of hydrogen peroxide and dimethyl sulfide borane poses an explosion risk and is environmentally unfriendly.

[0007] A proposed enzymatic resolution route was reported by [authors' name missing]. Dimethyl propylmalonate was subjected to strong base dehydrogenation, followed by reaction with tert-butyl bromoacetate, and decarboxylation to yield the enzyme-catalyzed substrate. Resolution was catalyzed by protease C from Bacillus subtilis. The enzyme-resolved product underwent esterification with ethyl chloroacetate, followed by reduction and acid cyclization to give (R)-4-propyldihydrofuran-2(3H)-one. This route achieved a resolution yield of 42% and exhibited poor atom economy. MERSCHAERT A,SZCZEPANIAK C,et al.A Biocatalytic Route to the Novel Antiepileptic Drug Brivaracetam[J].Organic Process Research&Development,2016,20(9):1566-1575.).

[0008] In recent years, methods for synthesizing buvastan intermediates using enzymatic asymmetric hydrogenation have been reported. CN107604018A discloses a method for synthesizing (R)-4-propyldihydrofuran-2-one from a substrate catalyzed by olefin reductase, but does not disclose information such as the olefin reductase sequence. Feng Jiacheng and Sun Lili et al. have also reported methods for preparing (R)-4-propyldihydrofuran-2-one using olefin reductase, but the substrate concentrations were only 7 g / L and 2.5 g / L, respectively (Process Biochemistry 126(2023)108–116; Applied Microbiology and Biotechnology(2023)107:1649–1661). In addition, patents such as CN111154735A, CN109852644A, CN 116948997 A and CN 118028391 A also disclose methods for preparing buvastan intermediates by chemical enzymatic methods, but these reports have disadvantages such as low substrate concentration, complicated operation and low conversion rate, which are not suitable for industrial production.

[0009] Therefore, there is an urgent need in this field to develop an environmentally friendly, efficient, highly stereoselective method for preparing the buvastan intermediate (R)-4-propyl-dihydrofuran-2-one that is more suitable for industrial production. Summary of the Invention

[0010] In view of the shortcomings of the prior art, the present invention provides an olefin reductase mutant and its use in catalyzing the preparation of buvastan synthetic intermediates, which can realize the efficient and green preparation of buvastan intermediate (R)-4-propyl-dihydrofuran-2-one.

[0011] In a first aspect, the present invention provides an olefin reductase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] Secondly, the present invention provides a nucleic acid encoding the aforementioned olefin reductase mutant; preferably, its nucleotide sequence is shown in SEQ ID NO.4.

[0013] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid.

[0014] Fourthly, the present invention provides a recombinant strain containing the aforementioned recombinant expression vector.

[0015] Fifthly, the present invention provides a complex enzyme comprising an olefin reductase mutant, a carbonyl reductase, and a glucose dehydrogenase, the amino acid sequences of which are respectively shown in SEQ ID NO. 1 to 3.

[0016] In a sixth aspect, the present invention provides a nucleic acid composition comprising: a first nucleic acid encoding the aforementioned alkene reductase mutant; a second nucleic acid encoding the aforementioned carbonyl reductase; and a third nucleic acid encoding the aforementioned glucose dehydrogenase; preferably, the nucleotide sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid are as shown in SEQ ID NO. 4 to 6, respectively.

[0017] In a seventh aspect, the present invention provides a recombinant expression vector composition comprising: a first recombinant expression vector comprising the aforementioned first nucleic acid; a second recombinant expression vector comprising the aforementioned second nucleic acid; and a third recombinant expression vector comprising the aforementioned third nucleic acid.

[0018] Eighthly, the present invention provides a recombinant strain composition comprising: a first recombinant strain comprising the aforementioned first recombinant expression vector; a second recombinant strain comprising the aforementioned second recombinant expression vector; and a third recombinant strain comprising the aforementioned third recombinant expression vector.

[0019] In a ninth aspect, the present invention provides the use of the aforementioned olefin reductase mutant, or nucleic acid, or recombinant expression vector, or recombinant strain, or complex enzyme, or nucleic acid composition, or recombinant expression vector composition, or recombinant strain composition in a biocatalyst for catalyzing the synthesis of (R)-4-propyldihydrofuran-2(3H)-one from 5-hydroxy-4-n-propyl-2-furanone.

[0020] In a tenth aspect, the present invention provides a biocatalytic preparation method of (R)-4-propyldihydrofuran-2(3H)-one, comprising: using the aforementioned composite enzyme or recombinant strain composition as a catalyst, and using 5-hydroxy-4-n-propyl-2-furanone as a substrate for biocatalysis; and, performing diastereomeric crystallization of the biocatalytic product to obtain (R)-4-propyldihydrofuran-2(3H)-one.

[0021] The biocatalysis stage also includes the use of reaction aids, including but not limited to: 1) nicotinamide adenine dinucleotide phosphate, glucose and phosphate buffer system; 2) nicotinamide adenine dinucleotide phosphate, isopropanol and phosphate buffer system; and 3) nicotinamide adenine dinucleotide phosphate, ammonium formate and phosphate buffer system.

[0022] Diastereomer crystallization involves first forming diastereomers with a chiral compound and a racemic mixture, then utilizing the solubility differences between the diastereomers to remove unwanted diastereomers through crystallization, thus achieving resolution. Preferably, the diastereomers are obtained by reacting 4-propyldihydrofuran-2(3H)-one with (S)-phenylethylamine, and then recrystallizing to remove the isomers.

[0023] The beneficial effects of this invention are as follows:

[0024] The present invention provides a complex enzyme composed of a mutant M8 of olefin reductase E1, carbonyl reductase K1, and glucose dehydrogenase. This enzyme can reduce the double bond and remove the hydroxyl group of compound 5-hydroxy-4-n-propyl-2-furanone as a substrate to obtain compound (R)-4-propyldihydrofuran-2(3H)-one. Compared with the use of unmutated olefin reductase E1, the ee value of the catalyzed (R)-4-propyldihydrofuran-2(3H)-one is increased from 39.30% to 93.0%. Based on this, diastereomeric crystallization yields (R)-4-propyldihydrofuran-2(3H)-one with an ee value >99.8%. This can be used for the one-pot biocatalytic preparation of key intermediates of buvasitial, reducing production costs, the use of polluting chemicals, and the risk of environmental pollution. Attached Figure Description

[0025] Figure 1 shows the gas phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one prepared by a one-pot method using olefin reductase E1-M8 and carbonyl reductase K1.

[0026] Figure 2 shows the chiral liquid phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one prepared by a one-pot method using olefin reductase E1-M8 and carbonyl reductase K1.

[0027] Figure 3 shows the chiral liquid phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one after purification by (S)-phenylethylamine crystallization. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Construction and expression of engineered olefin reductase bacteria

[0030] Prepare the culture medium using deionized water according to the following formula, sterilize at 121℃ for 20 minutes, and set aside for use.

[0031] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0032] Fermentation medium: yeast extract 24 g / L, soybean peptone 12 g / L, sodium chloride 3 g / L, glycerol 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L.

[0033] As shown in Table 1, genes derived from Bacillus halotolerans, Gluconobacter oxydans, Streptomyces sp.WP-1, Streptomyces albidoflavus, Saccharomyces cerevisiae S288C, Saccharomyces pastorianus, and Pseudomonas putida were synthesized after codon optimization, and then cloned into the pET22b(+) vector, which was then introduced into host Escherichia coli BL21(DE3) competent cells. After being cultured on ampicillin-resistant plates, single colonies were picked and cultured in LB medium to obtain recombinant genetically engineered bacteria with olefin reductases E1, E2, E3, E4, E5, E6, E7, and E8, respectively.

[0034] Recombinant genetically engineered bacteria of types E1, E2, E3, E4, E5, E6, E7, and E8 were transferred to 5 mL of LB medium (containing 50 μg / mL ampicillin) and cultured overnight at 37°C and 220 rpm to obtain seed culture. 10 μL of the seed culture was transferred to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 220 rpm. When the OD600 value > 0.8, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM to induce the expression of olefin reductases E1, E2, E3, E4, E5, E6, E7, and E8 at 25°C, and cultured overnight. After fermentation, the bacterial cells were collected by centrifugation at 12000 g for 10 min.

[0035] Table 1. Enyl reductases from different sources

[0036] Example 2: Construction and expression of engineered bacteria for glucose dehydrogenase

[0037] The glucose dehydrogenase GDH gene (accession number: WP_012369122.1) from *Exiguobacterium artemiae* was synthesized after codon optimization, cloned into the pET22b(+) vector, and then introduced into *E. coli* BL21(DE3) competent cells. The cells were cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB medium to obtain the recombinant genetically engineered glucose dehydrogenase strain. The strain was then fermented using the same method as in Example 1 above for later use.

[0038] Example 3: Preparation of enzyme-catalyzed substrate 5-hydroxy-4-n-propyl-2-furanone

[0039] Add 400 mL of heptane and 128 g of morpholine to a three-necked reaction flask, stir and mix, cool to 4 °C in an ice-water bath, and add 200 g of 50% glyoxylic acid aqueous solution dropwise, controlling the dropping rate and maintaining the temperature below 32 °C. After the addition is complete, react at room temperature for 1 h. Cool in an ice bath, and add 125 g of pentanal, controlling the internal temperature below 40 °C during the addition. After the addition is complete, react at 40 °C for 20 h, then cool to room temperature. Add 240 g of concentrated hydrochloric acid to adjust the pH of the aqueous layer to below 5. Separate the layers, wash the aqueous phase with heptane (500 mL × 2), extract with isopropyl ether (1000 mL × 3), separate the layers, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 134 g of a brownish-red oily substance, with a molar yield of 88.1%.

[0040] Example 4: Screening of olefin reductases

[0041] Prepare eight reaction solutions as follows: 10 g / L 5-hydroxy-4-n-propyl-2-furanone, 0.02 g / L NADP. +20 g / L glucose, 20 g / L glucose dehydrogenase, and 2 mL phosphate buffer (pH = 7.0) were added. Then, 50 g / L olefin reductases E1, E2, E3, E4, E5, E6, E7, and E8 were added to the reaction solution. The reaction was carried out at 220 rpm and 30 °C for 6 h. The conversion rate of substrate 4 to substrate 5 and the chirality of product 1 were measured, as shown in Table 2. E1 exhibited the best activity and chirality, with a conversion rate of 75.2% from substrate 4 to substrate 5 and 9.1% from substrate 4 to substrate 1, resulting in a product ee value of 39.3%.

[0042] Table 2. Reduction of 5-hydroxy-4-n-propyl-2-furanone by different olefin reductases

[0043] Gas chromatography detection method for enzyme-catalyzed reaction solution: Column: DB-5 30m×0.32mm×1.0μm; Injector temperature: 250℃; Detector temperature: 250℃; Column oven: 100℃ for 5 minutes, then increased to 250℃ at 10℃ / min and held for 5 minutes; Column flow rate: 2mL / min; Split ratio: 10:1; Injection volume: 1μl; Sample preparation: Take an appropriate amount of sample, dissolve and dilute with methanol to a 10mg / mL solution.

[0044] Chiral liquid chromatography detection method for enzyme-catalyzed products: Column: Phenomen Chiral NX(2) 5μm; Mobile phase: 90% n-hexane and 10% isopropanol; Column temperature: 30℃; Detection wavelength: 210nm; Flow rate: 0.5mL / min.

[0045] Example 5 Construction and screening of carbonyl reductase

[0046] Based on the results of Example 4, it was found that a large amount of 5-hydroxy-4-n-propyl-2-furanone substrate could only be converted to compound 5. In order to accelerate the conversion of 5-hydroxy-4-n-propyl-2-furanone to the final product 5-hydroxy-4-n-propyl-2-furanone (1), we explored some carbonyl reductases.

[0047] Genes derived from Nakaseomyces glabratus, Novosphingobium aromaticivorans DSM12444, Exiguobacterium acetylicum, and Meyerozyma guilliermondii ATCC 6260 were synthesized after codon optimization, then cloned into the pET22b(+) vector, and introduced into host Escherichia coli BL21(DE3) competent cells. After being cultured on ampicillin-resistant plates, single colonies were picked and cultured in LB medium to obtain recombinant genetically engineered bacteria containing carbonyl reductases K1, K2, K3, K4, and K5.

[0048] Glycerol-containing bacteria of the recombinant genetically engineered strains K1, K2, K3, K4, and K5 were transferred to 5 mL of LB medium (containing 50 μg / mL ampicillin) and cultured overnight at 37°C and 220 rpm to obtain seed culture. 10 μL of the seed culture was transferred to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 220 rpm. When the OD600 value > 0.8, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2 mM to induce the expression of carbonyl reductases K1, K2, K3, and K4 at 25°C, and cultured overnight. After fermentation, the bacterial cells were collected by centrifugation at 12000 g for 10 min.

[0049] Prepare six reaction solutions as follows: 10 g / L 5-hydroxy-4-n-propyl-2-furanone, 0.02 g / L NADP. + The reaction mixture consisted of 20 g / L glucose, 50 g / L olefin reductase E1, 25 g / L glucose dehydrogenase, and 2 mL phosphate buffer (pH = 7.0). Then, 50 g / L carbonyl reductases K1, K2, K3, K4, and K5 were added to the reaction solution. The reaction was carried out at 220 rpm and 30 °C for 6 h. The conversion rate and product chirality were then measured. The results are shown in Table 3. The combination of olefin reductase E1 and carbonyl reductase K1 showed the best conversion rate, with a substrate conversion rate of 100% and a product ee value of 39.3%.

[0050] Table 3. Catalytic reduction of 5-hydroxy-4-n-propyl-2-furanone by E1 in combination with different carbonyl reductases at a substrate concentration of 10 g / L.

[0051] Example 6: Construction and Screening of an Enylene Reductase Mutant Library

[0052] The protein structure of wild-type olefin reductase E1 was modeled using AlphaFold2, and molecular docking was performed between E1 and 5-hydroxy-4-n-propyl-2-furanone to select the distance from the substrate. Site-directed saturation mutagenesis and combinatorial saturation mutagenesis were performed on a range of amino acids. The constructed mutant plasmid library was transformed into *E. coli* BL21(DE3) and plated onto LB agar containing 50 μg / mL ampicillin, and incubated overnight at 37°C. Single colonies were picked and transferred to 96-well plates containing 400 μL of LB agar (containing 50 μg / mL ampicillin), and incubated overnight at 37°C and 200 rpm to obtain the mutant seed culture of olefin reductase E1. 10 μL of the mutant seed culture was transferred to a 96-well plate containing 400 μL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm until the OD600 value > 0.8. The mutant expression was induced with 1 mM isopropyl thiogalactoside (IPTG) at 28°C, followed by 20 h of further culture. The 96-well plate was then centrifuged at 4000 g for 30 min to collect the cells, followed by 200 μL of lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 10). 7.0) Resuspend the bacterial cells, then lyse them at 30°C for 1 hour. Centrifuge at 4°C, 4000g for 30 minutes, and collect the clear supernatant to determine the mutant activity. Detect the change in NADPH at 340nm using 190μL of the reaction solution (containing 0.5m / μL of NADPH). Calculate the enzyme activity of the mutants based on the NADPH consumption. Repeat the test with 2mL of the mutant solution. After fermentation of the mutant strain according to Example 1, react with carbonyl reductase K1. The reaction system was prepared according to Example 5. The conversion rate and chirality were measured after 3 hours of reaction. The chirality and relative activity of each mutant are shown in Table 4.

[0053] Table 4. Alkenyl reductase mutants and their relative activities Note: The activity of wild-type olefin reductase E1 is set to 100%.

[0054] As shown in Table 4, I69T is the site of the alkene reductase activity-enhancing mutant residue, with a relative activity of over 500%.

[0055] Example 7: Biocatalytic preparation of (R)-4-propyldihydrofuran-2(3H)-one

[0056] Add 0.5g of olefin reductase E1 mutant M8, 0.1g of 5-hydroxy-4-n-propyl-2-furanone, and 1mg of NADP to the reaction flask. + 0.5 g carbonyl reductase K1, 0.5 g glucose, 0.25 g glucose dehydrogenase, and 10 mL phosphate buffer (pH = 7.0) were added and reacted in a shaker at 220 rpm for 8 hours, with the pH maintained between 7.0 and 8.0. The substrate conversion was 100%, and the product ee value was 93.0%.

[0057] Example 8: Biocatalytic preparation of (R)-4-propyldihydrofuran-2(3H)-one

[0058] One-pot preparation: Add 45g of E1 mutant M8 cells, 45g of carbonyl reductase K1 cells, 27g of glucose dehydrogenase cells, and 0.54g of NADP to a 2L three-necked flask. + 27g of glucose and 90g of 5-hydroxy-4-n-propyl-2-furanone were added to 900mL of phosphate buffer solution and mechanically stirred until homogeneous. The mixture was heated in a water bath at 30℃. The pH was maintained between 7 and 8 throughout the reaction. After 12 hours of reaction, 1L of dichloromethane was added twice to extract the product, followed by mechanical stirring for 1 hour. The bacterial cells were filtered, and the organic phase was separated. The organic phase was washed twice with 5% hydrochloric acid, dried, and concentrated to obtain 73.9g of a yellow oily substance, with a molar yield of 91.1%, a purity of approximately 96%, and an ee of 93.0%. The gas chromatography and chiral liquid chromatography spectra are shown in Figure 1 and Figure 2, respectively.

[0059] Diastereomer crystallization: The yellow oily substance was reacted with (S)-phenylethylamine under alkaline conditions to give (R)-3-(hydroxymethyl)-N-((S)-1-phenylethyl)hexamethyleneamide. Tertiary methyl ether was added, the mixture was heated to dissolve, and then cooled to crystallize. The resulting solid was treated with concentrated sulfuric acid to remove (S)-phenylethylamine and then ring-closed (after removing phenylethylamine, the structure is chain-like, which requires acid to close the ring to obtain a five-membered ring lactone structure) to give the buvacertam intermediate (R)-4-propyldihydrofuran-2(3H)-one. The product ee > 99.8%, and the chiral liquid chromatography spectrum is shown in Figure 3.

[0060] Table 5 Sequence Information

[0061] Sequence 1 (SEQ ID NO: 1): MARKLFTPITIKDVTLKNRIVMSPMGMYSSHEKDGKLQPFHMAHYITRAVGQVGLIIVEASAVNPQGRTTDQDLGIWGDEHIEGFAKLTEQVKAQGSKIGIQLAHAGRKAELEGDIFAPSAIAFDEQSSTPVEMTTEKVKETVQEFKQAAARAKEAGFDVIEIHAA HGYLIHEFLSPLSNHRTDEYGGSPENRYRFLREIIDEVKQVWDGPLFVRVSASDYTDKGLDIADHIGFAKWMKEQGVDLIDCSSSGALVQADINVFPGYQVSFAEKIREQADMATGAVGMITNGSMAEEILQNNRADLIFIGRELLRDPYFARTAAKQLNTDIQAPVQYERGW*

[0062] Sequence 2 (SEQ ID NO: 2): MGSSHHHHHHSSGLVPRGSHMTTVFVSGATGFIAQHVVRQLLDQNYKVIGSVRSAEKGDHLKNVIFKGGDFNYEIVKDISDPTAFDHVFEKHGKDIKVVLHTASPFHFNTTDIEKDLLIPAVNGTKGILESIKKYAAQTVERVVVTSSFAANTSTVDMFYAKDSSKTITEESWNQDTWESCQSDPIRGYCGSKKFAEKAAWDFYNANKDSVKFKLSIINPVYVFGPQNYVEPGKKILNTSSEVINSLVHLKKDDPLPEFAGGHIDVRDVAKAHILAFQKDELIEQRLMLHAGLFTTQTLLDIINEQFPELKGKIPAGKPGTGNPDDALTPVDNSKTKKLLGFEFIDLKKDLYDTISQILEAEKNSN

[0063] Sequence 3 (SEQ ID NO: 3): MHHHHHHYNSLKGKVAIVTGGSMGIGEAIIRRYAEEGMRVVINYRSHPEEAKKIAEDIKQAGGEALTVQGDVSKEEDMINLVKQTVDHFGQLDVFVNNAGVEMPSPSHEMSLEDWQKVIDVNLTGAFLGAREALKYFVEHNVKGNIINMSSVHEIIPWPTFVHYAASKGGVKLMTQTLAMEYAPKGIRINAIGPGAINTPINAEKFEDPKQRADVESMIPMGNIGKPEEISAVAAWLASDEASYVTGITLFADGGMTLYPSFQAGRG*

[0064]

[0065]

[0066] Sequence 6 (SEQ ID NO: 6): ATGCATCATCATCATCATCACTATAATTCACTAAAGGGAAAAGTAGCTATTGTTACCGGCGGCAGCATGGGTATCGGCGAAGCGATTATTCGCCGTTATGCCGAGGAGGGTATGCGTGTTGTGATCAATTATCGTAGCCATCCAGAAGAAGCTAAAAAAATCGCGGAGGACATCAAACAAGCAGGCGGCGAGGCGC TGACCGTACAGGGTGATGTTAGCAAAGAAGAGGACATGATTAACCTGGTCAAGCAAACTGTCGATCATTTTGGTCAGCTGGATGTTTTTGTCAACAACGCGGGCGTGGAGATGCCGTCGCCGTCCCATGAAATGAGCCTGGAAGACTGGCAGAAGGTGATTGATGTGAATTTGACGGGTGCCTTCCTGGGTGCGCGTGAAGCC TTAAAATACTTCGTGGAGCACAACGTGAAGGGCAACATCATCAACATGTCAAGCGTTCACGAGATCATTCCGTGGCCTACATTCGTGCACTACGCCGCTTCCAAGGGTGGCGTTAAGCTGATGACCCAGACGCTCGCTATGGAATACGCTCCGAAGGGCATCCGCATTAACGCGATCGGTCCGGGTGCGATCAACACCCGA TTAATGCGGAGAAATTCGAAGACCCGAAGCAACGCGCAGACGTGGAGAGCATGATCCCGATGGGCAATATTGGTAAACCGGAGGAGATTAGCGCAGTTGCAGCGTGGCTTCTGACGAAGCGTCCTATGTTACCGGGATCACCTTGTTTGCAGATGGTGGCATGACCCTGTACCCGTCTTTTCAGGCGGGTCGTGGTTAA

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A olefin reductase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that, It encodes the olefin reductase mutant of claim 1.

3. The nucleic acid according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 2 or 3.

5. A recombinant bacterial strain, characterized in that, It contains the recombinant expression vector as described in claim 4.

6. A complex enzyme, characterized in that, It contains olefin reductase mutants, carbonyl reductase and glucose dehydrogenase, with amino acid sequences as shown in SEQ ID NO.1-3, respectively.

7. A nucleic acid composition, characterized in that, The nucleic acid composition comprises: First nucleic acid: Encoding the olefin reductase mutant as described in claim 6; Second nucleic acid: encoding the carbonyl reductase as described in claim 6; and Third nucleic acid: Encodes the glucose dehydrogenase as described in claim 6.

8. The nucleic acid composition according to claim 7, characterized in that, The nucleotide sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid are shown in SEQ ID NO.4 to 6, respectively.

9. A recombinant expression vector composition comprising: First recombinant expression vector: comprising the first nucleic acid as described in claim 7; Second recombinant expression vector: comprising the second nucleic acid as described in claim 7; and The third recombinant expression vector comprises the third nucleic acid as described in claim 7.

10. A recombinant bacterial strain composition comprising: First recombinant strain: comprising the first recombinant expression vector as described in claim 9; Second recombinant strain: comprising the second recombinant expression vector as described in claim 9; and Third recombinant strain: comprising the third recombinant expression vector as described in claim 9.

11. The use of the olefin reductase mutant of claim 1, or the nucleic acid of any one of claims 2-3, or the recombinant expression vector of claim 4, or the recombinant strain of claim 5, or the complex enzyme of claim 6, or the nucleic acid composition of any one of claims 7-8, or the recombinant expression vector composition of claim 9, or the recombinant strain composition of claim 10, in a biocatalyst for the synthesis of (R)-4-propyldihydrofuran-2(3H)-one from 5-hydroxy-4-n-propyl-2-furanone.

12. A biocatalytic preparation method for (R)-4-propyldihydrofuran-2(3H)-one, characterized in that, The preparation method includes: Using the complex enzyme of claim 6 or the recombinant bacterial composition of claim 10 as a catalyst, biocatalysis is performed with 5-hydroxy-4-n-propyl-2-furanone as a substrate; and, The biocatalytic product was subjected to diastereomeric crystallization to obtain (R)-4-propyldihydrofuran-2(3H)-one.

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