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

Abstract

The invention belongs to the technical field of biocatalysts, and discloses an alkene reductase mutant and application of the alkene reductase mutant in catalytic preparation of a brivaracetam synthesis intermediate. The amino acid sequence of the alkene reductase mutant is as shown in SEQ ID NO.1, a compound enzyme composed of the mutant, carbonyl reductase K1 and glucose dehydrogenase can take a compound 5-hydroxy-4-n-propyl-2-furanone as a substrate, double bonds are reduced, hydroxyl is removed, a compound (R)-4-propyl dihydrofuran-2 (3H)-ketone is obtained, and the compound (R)-4-propyl dihydrofuran-2 (3H)-ketone can be used for preparing the alkene reductase. The ee value of the (R)-4-propyl dihydrofuran-2 (3H)-ketone obtained through catalysis reaches 93.0%, the (R)-4-propyl dihydrofuran-2 (3H)-ketone with the ee value larger than 99.8% can be further obtained through diastereoisomer crystallization, and the method can be used for efficient and green preparation of the brivaracetam key intermediate (R)-4-propyl dihydrofuran-2 (3H)-ketone.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysts, and particularly relates to an alkene reductase mutant and an application of the mutant in catalyzing the preparation of a brivaracetam synthetic intermediate. Background Art

[0002] Brivaracetam, also known as brivaracetam, has a chemical name of (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide, which is a structural analog of levetiracetam. Brivaracetam is a third-generation anti-epileptic drug developed by UC Pharma of Belgium. It can selectively bind to the central synaptic vesicle protein 2A, regulate the release of intersynaptic neurotransmitters and play an anti-epileptic role. It has the advantages of high bioavailability and good safety.

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

[0004]

[0005] Patent WO2016191435A1 uses (R)-epichlorohydrin as the starting material to introduce a chiral center, and obtains (R)-4-propyldihydrofuran-2(3H)-one through nucleophilic substitution reaction, Grignard reaction, and decarboxylation reaction. The raw materials used in this route are cheap and easy to obtain, but the reaction conditions of the Grignard reaction are harsh, and multiple vacuum distillation operations are required throughout the process. The ee of the product is 98%, which still needs to be further improved.

[0006]

[0007] Patent CN105801530 uses chiral raw material (R)-2-aminopentanoic acid, replaces it with hydrogen bromide to obtain (R)-2-bromovaleric acid, then uses diborane to reduce the carboxyl group, tert-butyldimethylsilyl protects the hydroxyl group, nucleophilic substitution, deprotection, hydrolysis, and esterification to obtain (R)-4-propyldihydrofuran-2(3H)-one. This route uses highly toxic hydrogen bromide and diborane, which has safety risks.

[0008]

[0009] Patent CN106008411A uses (S)-4-benzyl-2-oxazolidinone as a chiral auxiliary, reacts with n-pentanoic acid and pivaloyl chloride, and then reacts with tert-butyl bromoacetate under the action of a strong base, and then removes the auxiliary group oxazolidinone by hydrogen peroxide and lithium hydroxide. Finally, the carboxyl group is reduced and the ring is closed to obtain (R)-4-propyldihydrofuran-2(3H)-one. This route requires an ultra-low temperature of -70°C, and the reaction conditions are relatively harsh. The use of hydrogen peroxide and dimethyl sulfide borane has the risk of explosion and is not environmentally friendly.

[0010]

[0011] A et al. reported an enzymatic splitting route. Propylmalonate was used to extract hydrogen with a strong base, react with tert-butyl bromoacetate, and then decarboxylate to obtain an enzyme-catalyzed substrate. The enzyme splitting product was esterified with ethyl chloroacetate, and then reduced and acid-added to obtain (R)-4-propyldihydrofuran-2(3H)-one. The splitting yield of this route was 42%, and the atom economy was poor ( A,MERSCHAERTA,SZCZEPANIAK C,et al.ABiocatalytic Route to the Novel Antiepileptic DrugBrivaracetam[J].Organic Process Research&Development,2016,20(9):1566-1575.).

[0012] In recent years, methods for synthesizing buvacetam intermediates by enzymatic asymmetric hydrogenation have been reported one after another. CN107604018A discloses a method for synthesizing (R)-4-propyldihydrofuran-2-one from 4-n-propyl-2(5H)-furanone substrate catalyzed by ene reductase, but no information such as the ene reductase sequence is disclosed. Feng Jiacheng, Sun Lili et al. also reported a method for preparing (R)-4-propyldihydrofuran-2-one using ene 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, CN116948997A and CN 118028391 A also disclose methods for preparing brivaracetam intermediates by chemical enzymatic methods, but these reports have disadvantages such as low substrate concentration, complex operation and low conversion rate, and are not suitable for industrial production.

[0013] Therefore, there is an urgent need in the art to develop a method for preparing the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one that is environmentally friendly, efficient, highly stereoselective, and more suitable for industrial production. Summary of the invention

[0014] In view of the deficiencies in the prior art, the present invention provides an ene reductase mutant and its use in catalyzing the preparation of a brivaracetam synthetic intermediate, which can achieve efficient and green preparation of the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one.

[0015] 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.

[0016] In a second aspect, the present invention provides a nucleic acid encoding the aforementioned olefin reductase mutant; preferably, its nucleotide sequence is shown in SEQ ID NO.4.

[0017] In a third aspect, the present invention provides a recombinant expression vector comprising the aforementioned nucleic acid.

[0018] In a fourth aspect, the present invention provides a recombinant strain comprising the aforementioned recombinant expression vector.

[0019] In a fifth aspect, the present invention provides a composite enzyme comprising an olefin reductase mutant, a carbonyl reductase and a glucose dehydrogenase, the amino acid sequences of which are shown in SEQ ID NOs. 1 to 3, respectively.

[0020] In a sixth aspect, the present invention provides a nucleic acid composition comprising: a first nucleic acid encoding the aforementioned ene 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 respectively as shown in SEQ ID NO.4 to 6.

[0021] 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.

[0022] In an eighth aspect, 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.

[0023] In the ninth aspect, the present invention provides the use of the aforementioned ene 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 that catalyzes the synthesis of (R)-4-propyldihydrofuran-2(3H)-one from 5-hydroxy-4-n-propyl-2-furanone.

[0024] 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 5-hydroxy-4-n-propyl-2-furanone as a substrate for biocatalysis; and performing diastereoisomer crystallization on the biocatalytic product to obtain (R)-4-propyldihydrofuran-2(3H)-one.

[0025] The biocatalytic stage also includes the use of reaction aids, including but not limited to: 1) nicotinamide adenine dinucleoside phosphate, glucose and phosphate buffer system; 2) nicotinamide adenine dinucleoside phosphate, isopropanol, phosphate buffer system; and 3) nicotinamide adenine dinucleoside phosphate, ammonium formate, phosphate buffer system.

[0026] Diastereoisomer crystallization is to first use a chiral compound and a racemate to form diastereomers, and then use the difference in solubility between diastereomers to remove unwanted diastereomers by crystallization to achieve separation. Preferably, the diastereomers are obtained by reacting 4-propyldihydrofuran-2(3H)-one with (S)-phenylethylamine, and then recrystallized to remove the isomers.

[0027] The beneficial effects of the present invention are:

[0028] The composite enzyme composed of the mutant M8 of olefin reductase E1 provided by the present invention, carbonyl reductase K1 and glucose dehydrogenase can use the compound 5-hydroxy-4-n-propyl-2-furanone as a substrate, reduce double bonds and remove hydroxyl groups to obtain the compound (R)-4-propyldihydrofuran-2(3H)-one. Compared with the unmutated olefin reductase E1, the ee value of the catalytically obtained (R)-4-propyldihydrofuran-2(3H)-one is increased from 39.30% to 93.0%. On this basis, (R)-4-propyldihydrofuran-2(3H)-one with an ee value greater than 99.8% can be obtained by diastereoisomer crystallization. It can be used for the one-pot biocatalytic preparation of key intermediates of buvacetan, which reduces production costs, reduces the use of polluting chemicals, and reduces the risk of environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The gas phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one prepared in one pot using ene reductase E1-M8 and carbonyl reductase K1;

[0030] Figure 2 The chiral liquid phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one prepared in one pot using ene reductase E1-M8 and carbonyl reductase K1;

[0031] Figure 3 This is the chiral liquid phase spectrum of (R)-4-propyldihydrofuran-2(3H)-one after purification by (S)-phenylethylamine crystallization. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1 Construction and expression of olefin reductase engineering bacteria

[0034] Prepare the culture medium with deionized water according to the following formula, sterilize at 121°C for 20 min, and set aside.

[0035] LB liquid medium: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L.

[0036] Fermentation medium: yeast extract 24 g / L, soy 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.

[0037] 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 pET22b (+) vectors, and then introduced into host Escherichia coli BL21 (DE3) competent cells, cultured on ampicillin resistance plates, and single colonies were picked and cultured in LB medium, and finally recombinant genetically engineered bacteria of ene reductase E1, E2, E3, E4, E5, E6, E7 and E8 were obtained, respectively.

[0038] Pick the recombinant genetic engineering bacteria of E1, E2, E3, E4, E5, E6, E7 and E8 above to 5mL LB medium (containing 50μg / mL ampicillin), and culture overnight at 37°C and 220rpm to obtain seed liquid. Pipette 10μL seed liquid and transfer to a shake flask containing 50mL fermentation medium (containing 50μg / mL ampicillin), and culture at 37°C and 220rpm. When the OD600 value is >0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.2mM to induce the expression of ene reductase E1, E2, E3, E4, E5, E6, E7 and E8 at 25°C, and culture overnight. After the fermentation is completed, centrifuge at 12000g for 10min to collect the bacteria.

[0039] Table 1 Oleoreductase from different sources

[0040]

[0041]

[0042] Example 2 Construction and expression of glucose dehydrogenase engineering bacteria

[0043] The glucose dehydrogenase GDH gene from Exiguobacterium artemiae (accession number: WP_012369122.1) was synthesized after codon optimization, and then cloned into the pET22b (+) vector, and then introduced into the host Escherichia coli BL21 (DE3) competent cells, cultured on an ampicillin-resistant plate, and a single colony was picked and cultured in LB medium to finally obtain glucose dehydrogenase recombinant genetic engineering bacteria. Fermented in the same manner as in Example 1 above for later use.

[0044] Example 3 Preparation of Enzyme-Catalyzed Substrate 5-Hydroxy-4-n-propyl-2-furanone

[0045]

[0046] Add 400mL of heptane and 128g of morpholine to a three-necked reaction flask, stir and mix, cool to 4°C in an ice-water bath, and drop 200g of a 50% aqueous solution of glyoxylic acid, control the dropping speed, and keep the temperature below 32°C. After the drop is complete, react at room temperature for 1h. Cool in an ice bath, drop 125g of valeraldehyde, and control the internal temperature to be less than 40°C during the drop. After the drop is complete, react at 40°C for 20h and then cool to room temperature. Drop 240g of concentrated hydrochloric acid to adjust the pH of the water layer to below 5. Separate the liquid, wash the aqueous phase with heptane (500mL×2), extract with isopropyl ether (1000mL×3), separate the liquid, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 134g of a brown-red oil with a molar yield of 88.1%.

[0047] Example 4 Oleoreductase Screening

[0048]

[0049] Prepare 8 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, 2 mL phosphate buffer (pH = 7.0). Then 50 g / L ene reductase E1, E2, E3, E4, E5, E6, E7 and E8 were added to the reaction solution, respectively, and the reaction was carried out at 220 rpm, 30 ° C, and after 6 hours, the conversion rate of substrate 4 to 5 and the chirality of product 1 were determined, as shown in Table 2. Among them, E1 has the best activity and chirality, with a conversion rate of substrate 4 to 5 of 75.2%, a conversion rate of 4 to 1 of 9.1%, and a product ee value of 39.3%.

[0050] Table 2 Reduction of 5-hydroxy-4-n-propyl-2-furanone catalyzed by different ene reductases

[0051]

[0052] Gas phase detection method for enzyme-catalyzed reaction liquid: chromatographic column: DB-530m×0.32mm×1.0μm; inlet temperature: 250℃, detector temperature: 250℃; column oven: 100℃ for 5 minutes, increase to 250℃ at 10℃ / min, and maintain for 5 minutes; column flow rate: 2mL / min; split ratio: 10:1; injection volume 1μl; sample preparation: take an appropriate amount of sample, dissolve it with methanol and dilute it to a 10mg / mL solution.

[0053] Chiral liquid phase detection method for enzyme-catalyzed products: chromatographic column: Phenol Chiral NX (2) 5μ; mobile phase: 90% n-hexane and 10% isopropanol; column temperature: 30°C; detection wavelength: 210nm; flow rate: 0.5mL / min.

[0054] Example 5 Construction and screening of carbonyl reductase

[0055] According to 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.

[0056] Genes from Nakaseomyces glabratus, Novosphingobium aromaticivorans DSM12444, Exiguobacterium acetylicum and Meyerozyma guilliermondii ATCC 6260 were synthesized after codon optimization, and then cloned into pET22b(+) vectors, and then introduced into host Escherichia coli BL21(DE3) competent cells, cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB medium, and finally recombinant genetically engineered bacteria of carbonyl reductase K1, K2, K3, K4 and K5 were obtained, respectively.

[0057] Pick the glycerol bacteria of the recombinant genetic engineering bacteria of K1, K2, K3, K4 and K5 above to 5mL LB medium (containing 50μg / mL ampicillin), and culture overnight at 37°C and 220rpm to obtain seed liquid. Pipette 10μL of seed liquid and transfer to a shake flask containing 50mL fermentation medium (containing 50μg / mL ampicillin), and culture at 37°C and 220rpm. When the OD600 value is >0.8, add isopropylthiogalactoside (IPTG) at a final concentration of 0.2mM to induce the expression of carbonyl reductase K1, K2, K3 and K4 at 25°C, and culture overnight. After the fermentation is completed, centrifuge at 12000g for 10min to collect the bacteria.

[0058]

[0059] Prepare 6 reaction solutions as follows: 10 g / L 5-hydroxy-4-n-propyl-2-furanone, 0.02 g / L NADP + , 20g / L glucose, 50g / L ene reductase E1, 25g / L glucose dehydrogenase, 2mL phosphate buffer (pH=7.0). Then 50g / L carbonyl reductase K1, K2, K3, K4 and K5 were added to the reaction solution respectively, and the reaction was carried out at 220rpm, 30℃, and the conversion rate and product chirality were determined. The results are shown in Table 3, among which, the conversion rate of ene reductase E1 combined with carbonyl reductase K1 was the best, the substrate conversion rate was 100%, and the product ee value was 39.3%.

[0060] Table 3 Catalytic reduction of 5-hydroxy-4-n-propyl-2-furanone by E1 combined with different carbonyl reductases at 10 g / L substrate concentration

[0061]

[0062]

[0063] Example 6 Construction and screening of olefin reductase mutant library

[0064] AlphaFold2 was used to model the protein structure of wild-type ene reductase E1, and 5-hydroxy-4-n-propyl-2-furanone was used for molecular docking with E1 to select the distance to the substrate. The constructed mutant plasmid library was transferred into Escherichia coli BL21 (DE3), spread on LB solid medium containing 50 μg / mL ampicillin, and cultured overnight in a 37°C incubator. A single colony was picked into a 96-well plate containing 400 μL LB medium (containing 50 μg / mL ampicillin), and cultured overnight at 37°C, 200 rpm to obtain the mutant seed solution of ene reductase E1. 10 μL of mutant seed solution was transferred to a 96-well plate containing 400 μL fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm until the OD600 value was >0.8. The mutant was induced to express using isopropylthiogalactoside (IPTG) at a final concentration of 1 mM at 28°C, and then cultured for 20 h. The 96-well plate was placed in a centrifuge and centrifuged at 4000 g for 30 min to collect the cells, and then lysed with 200 μL of lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 5.0). 7.0) resuspend the cells, then lyse at 30°C for 1h, centrifuge at 4°C, 4000g, centrifuge for 30min, and draw the clarified supernatant to determine the activity of the mutant. 190μL of the reaction solution (containing 0.5m / 340nm of light to detect the change of NADPH. The consumption of NADPH is used to calculate the enzyme activity of the reaction mutant. Pick a part of the mutant and add 2mL to the reaction for re-testing. After the mutant strain is fermented according to Example 1, carbonyl reductase K1 is used in combination for reaction. The reaction system is prepared according to Example 5. The conversion rate and chirality are measured after 3h of reaction. The chirality and relative activity of each mutant are shown in Table 4.

[0065] Table 4 Oleoreductase mutants and their relative activities

[0066]

[0067] Note: The activity of wild-type ene reductase E1 was set as 100%.

[0068] As shown in Table 4, I69T is the activity enhancement sensitive mutation residue position of olefin reductase, and the relative activity can reach more than 500%.

[0069] Example 7 Biocatalytic Preparation of (R)-4-Propyldihydrofuran-2(3H)-one

[0070] Add 0.5 g of mutant M8 of ene reductase E1, 0.1 g of 5-hydroxy-4-n-propyl-2-furanone, and 1 mg of NADP to the reaction bottle. +, 0.5g carbonyl reductase K1, 0.5g glucose, 0.25g glucose dehydrogenase, 10mL phosphate buffer (pH=7.0), react in a shaker for 8h, speed 220rpm, during which pH=7.0-8.0 was controlled. Substrate conversion rate was 100%, and product ee value was 93.0%.

[0071] Example 8 Biocatalytic Preparation of (R)-4-Propyldihydrofuran-2(3H)-one

[0072] One-pot preparation: Add 45g E1 mutant M8 bacteria, 45g carbonyl reductase K1 bacteria, 27g glucose dehydrogenase bacteria, 0.54g NADP to a 2L three-necked flask. + , 27g glucose, 90g 5-hydroxy-4-n-propyl-2-furanone, add 900mL phosphate buffer solution and stir mechanically. Heat in a water bath, internal temperature 30℃. The pH is always controlled at 7-8 during the reaction. After 12h of reaction, add 1L of dichloromethane to extract the product twice, stir mechanically for 1h, filter the bacteria, separate the liquid to obtain the organic phase, wash twice with 5% hydrochloric acid, dry and concentrate the organic phase, and obtain 73.9g of yellow oil, with a molar yield of 91.1%, a purity of about 96%, an ee of 93.0%, and gas phase spectrum and chiral liquid phase spectrum as shown respectively. Figure 1 , Figure 2 shown.

[0073] Diastereoisomer crystallization: The yellow oil is reacted with (S)-phenylethylamine under alkaline conditions to obtain (R)-3-(hydroxymethyl)-N-((S)-1-phenylethyl)hexanamide, methyl tert-ether is added, the temperature is raised to dissolve, the temperature is lowered to crystallize, and the obtained solid is removed by concentrated sulfuric acid to remove (S)-phenylethylamine and ring-close (a chain structure is formed after the removal of phenylethylamine, and acid needs to be added to close the ring to obtain a five-membered ring lactone structure) to obtain the buvacetam intermediate (R)-4-propyldihydrofuran-2(3H)-one, the product ee>99.8%, and the chiral liquid phase spectrum is as follows Figure 3 shown.

[0074] Table 5 Sequence information

[0075] Sequence number (SEQ ID NO:) describe 1 Amino acid sequence of mutant M8 of olefin reductase E1 2 Amino acid sequence of carbonyl reductase K1 3 Amino acid sequence of glucose dehydrogenase GDH 4 Nucleotide sequence of mutant M8 of olefin reductase E1 5 Nucleotide sequence of carbonyl reductase K1 6 Nucleotide sequence of glucose dehydrogenase GDH

[0076] Sequence 1 (SEQ ID NO: 1): MARKLFTPITIKDVTLKNRIVMSPMGMYSSHE KDGKLQPFHMAHYITRAVGQVGLIIVEASAVNPQGRTTDQDLGIWGDEHIEGFAKLTEQVKAQGSKIGIQLAHAGRKAELEGDIFAPSAIAFDEQSSTPVEMTTEKVKETVQEFKQAAARAKEAGFDVIEIHAAHGYLIHEFLSPLSNHRTDEYGGSPENRYRFLREIIDEVKQVWDGPLFVRVSASDYTDKGLDIADHIGFAKWMKEQGVDLIDCSSGALVQADINVFPGYQVSFAEKIREQADMATGAVGMITNGSMAEEILQNNRADLIFIGRELLRDPYFARTAAKQLNTDIQAPVQYERGW*

[0077] Sequence 2 (SEQ ID NO: 2): MGSSHHHHHHSSGLVPRGSHMTTVFVSGATG FIAQHVVRQLLDQNYKVIGSVRSAEKGDHLKNVIFKGGDFNYEIVKDISDPTAFDHVFEKHGKDIKVVLHTASPFHFNTTDIEKDLLIPAVNGTKGILESIKKYAAQTVERVVVTSSFAANTSTVDMFYAKDSSKTITEESWNQDTWESCQSDPIRGYCGSKKFAEKAAWDFYNANKDSVKFKLSIINPVYVFGPQNYVEPGKKILNTSSEVINSLVHLKKDDPLPEFAGGHIDVRDVAKAHILAFQKDELIEQRLMLHAGLFTTQTLLDIINEQFPELKGKIPAGKPGTGNPDDALTPVDNSKTKKLLGFEFIDLKKDLYDTISQILEAEKNSN

[0078] Sequence 3 (SEQ ID NO: 3): MHHHHHHYNSLKGKVAIVTGGSMGIGEAIIRR YAEEGMRVVINYRSHPEEAKKIAEDIKQAGGEALTVQGDVSKEEDMINLVKQTVDHFGQLDVFVNNAGVEMPSPSHEMSLEDWQKVIDVNLTGAFLGAREALKYFVEHNVKGNIINMSSVHEIIPWPTFVHYAASKGGVKLMTQTLAMEYAPKGIRINAIGPGAINTPINAEKFEDPKQRADVESMIPMGNIGKPEEISAVAAWLASDEASYVTGITLFADGGMTLYPSFQAGRG*

[0079] Sequence 4 (SEQ ID NO: 4): ATGGCGCGCAAACTGTTTACCCCGATTACCATTAAAGATGTGACCCTGAAAAACCGCATTGTGATGAGCCCGATGGGCATGTATAGCAGCCATGAAAAAGATGGCAAACTGCAGCCGTTTCACATGGCGCATTATATTACCCGCGCGGTGGGCCAAGTGGGCCTGATTATTGTGGAAGCGAGCGCGGTGAACCCGCAAGGCCGCACCACCGATCAAGATCTGGGCATTTGGGGCGATGAACATATTGAAGGCTTCGCGAAACTGACCGAACAAGTGAAAGCGCAAGGCAGCAAAATTGGCATTCAGCTGGCGCATGCGGGCCGCAAAGCGGAACTGGAAGGCGATATTTTTGCGCCGAGCGCGATTGCGTTTGATGAACAGAGCAGCACCCCGGTGGAAATGACCACCGAAAAAGTGAAAGAAACCGTGCAAGAATTTAAACAAGCGGCCGCGCGCGCGAAAGAAGCGGGCTTTGATGTGATTGAAATTCATGCGGCGCATGGCTATCTGATTCATGAATTTCTGAGCCCGCTGAGCAACCATCGCACCGATGAATATGGCGGCAGCCCGGAAAACCGCTATCGCTTTCTGCGCGAAATTATTGATGAAGTGAAACAAGTGTGGGATGGCCCGCTGTTTGTGCGCGTGAGCGCGAGCGATTATACCGATAAAGGCCTGGATATTGCGGATCATATTGGCTTTGCGAAATGGATGAAAGAACAAGGCGTGGATCTGATTGATTGCAGCAGCGGCGCGCTGGTGCAAGCGGATATTAACGTGTTTCCGGGCTATCAAGTGAGCTTTGCGGAAAAAATTCGCGAACAAGCGGATATGGCGACCGGCGCGGTGGGCATGATTACCAACGGCAGCATGGCGGAAGAAATTCTGCAGAACAACCGCGCGGATCTGATTTTTATTGGCCGCGAACTGCTGCGCGATCCGTATTTTGCGCGCACCGCGGCGAAACAGCTGAACACCGATATTCAAGCGCCGGTGCAGTATGAACGCGGCTGGTAA

[0080]

[0081] Sequence 6 (SEQ ID NO: 6): ATGCATCATCATCATCATCACTATAATTCACT AAAGGGAAAAGTAGCTATTGTTACCGGCGGCAGCATGGGTATCGGCGAAGCGATTATTCGCCGTTATGCCGAGGAGGGTATGCGTGTTGTGATCAATTATCGTAGCCATCCAGAAGAAGCTAAAAAAATCGCGGAGGACATCAAACAAGCAGGCGGCGAGGCGCTGACCGTACAGGGTGATGTTAGCAAAGAA GAGGACATGATTAACCTGGTCAAGCAAACTGTCGATCATTTTGGTCAGCTGGATGTTTTTGTCAACAACGCGGGCGTGGAGATGCCGTCGCCGTCCCATGAAATGAGCCTGGAAGACTGGCAGAAGGTGATTGATGTGAATTTGACGGGTGCCTTCCTGGGTGCGCGTGAAGCCTTAAAATACTTCGTGGAGC ACAACGTGAAGGGCAACATCATCAACATGTCAAGCGTTCACGAGATCATTCCGTGGCCTACATTCGTGCACTACGCCGCTTCCAAGGGTGGCGTTAAGCTGATGACCCAGACGCTCGCTATGGAATACGCTCCGAAGGGCATCCGCATTAACGCGATCGGTCCGGGTGCGATCAACACCCCGATTAATGCGGA GAAATTCGAAGACCCGAAGCAACGCGCAGACGTGGAGAGCATGATCCCGATGGGCAATATTGGTAAACCGGAGGAGATTAGCGCAGTTGCAGCGTGGTTGGCTTCTGACGAAGCGTCCTATGTTACCGGGATCACCTTGTTTGCAGATGGTGGCATGACCCTGTACCCGTCTTTTCAGGCGGGTCGTGGTTAA

[0082] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. An 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 according to 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 according to claim 2 or 3.

5. A recombinant strain, characterized in that: It contains the recombinant expression vector according to claim 4.

6. A composite enzyme, characterized in that The invention comprises an alkene reductase mutant, a carbonyl reductase and a glucose dehydrogenase, the amino acid sequences of which are shown in SEQ ID NOs. 1 to 3 respectively.

7. A nucleic acid composition, characterized in that The nucleic acid composition comprises: The first nucleic acid encodes the olefin reductase mutant according to claim 6; A second nucleic acid encoding the carbonyl reductase according to claim 6; and The third nucleic acid encodes the glucose dehydrogenase according to 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 NOs. 4 to 6, respectively.

9. A recombinant expression vector composition, comprising: A first recombinant expression vector comprising the first nucleic acid according to claim 7; A second recombinant expression vector comprising the second nucleic acid of claim 7; and The third recombinant expression vector comprises the third nucleic acid according to claim 7.

10. A recombinant strain composition comprising: A first recombinant strain comprising the first recombinant expression vector according to claim 9; A second recombinant strain comprising the second recombinant expression vector according to claim 9; and The third recombinant strain comprises the third recombinant expression vector according to claim 9.

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

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

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