Olefin reductase mutant and its use in catalyzing the preparation of brivaracetam synthetic intermediates

Abstract

The present invention belongs to the field of biocatalyst technology and discloses an alkene reductase mutant and its use in catalyzing the preparation of a brivaracetam synthetic intermediate. The amino acid sequence of the alkene reductase mutant of the present invention is shown in SEQ ID NO.1. The mutant, carbonyl reductase K1, and glucose dehydrogenase form a composite enzyme that 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. The catalytically obtained (R)-4-propyldihydrofuran-2(3H)-one has an ee value of 93.0%, and can further obtain (R)-4-propyldihydrofuran-2(3H)-one with an ee value greater than 99.8% through diastereoisomer crystallization. The complex enzyme can be used for the efficient and green preparation of (R)-4-propyldihydrofuran-2(3H)-one, a key intermediate of brivaracetam.

Description

Technical Field

[0001] The present 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 synthesis intermediate. Background Art

[0002] Brivaracetam, also known as brivaracetam, has the chemical name (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide and is a structural analog of levetiracetam. Developed by UC Pharma of Belgium, Brivaracetam is a third-generation anti-epileptic drug that selectively binds to central nervous system synaptic vesicle protein 2A, modulating the release of neurotransmitters between synapses and exerting its anti-epileptic effects. 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 minimal steric hindrance between the five-membered ring lactone intermediate and its enantiomer, its preparation and separation are challenging. Therefore, developing a new asymmetric catalytic synthesis method to obtain high-optical purity brivaracetam intermediates remains an urgent challenge.

[0004]

[0005] Patent WO2016191435A1 introduces a chiral center using (R)-epichlorohydrin as the starting material. (R)-4-propyldihydrofuran-2(3H)-one is obtained through nucleophilic substitution, Grignard reaction, and decarboxylation. This route utilizes inexpensive and readily available raw materials, but the Grignard reaction requires demanding conditions, requiring multiple vacuum distillations. The product's ee is only 98%, which remains to be improved.

[0006]

[0007] Patent CN105801530 uses a chiral raw material (R)-2-aminopentanoic acid, which is substituted with hydrogen bromide to obtain (R)-2-bromovaleric acid. The carboxyl group is then reduced with diborane, the hydroxyl group is protected with tert-butyldimethylsilyl, and nucleophilic substitution, deprotection, hydrolysis, and esterification are performed to obtain (R)-4-propyldihydrofuran-2(3H)-one. This route uses highly toxic hydrogen bromide and diborane, posing a safety risk.

[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 in the presence of a strong base. The auxiliary group oxazolidinone is then removed 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 resolution route. Propylmalonate was subjected to strong base hydrogenation, reacted with tert-butyl bromoacetate, and decarboxylated to obtain the enzymatic substrate. The enzymatic resolution was catalyzed by proteinase C from Bacillus subtilis. The enzymatic resolution product was esterified with ethyl chloroacetate, and then reduced and acid-added to give (R)-4-propyldihydrofuran-2(3H)-one. This route yielded 42% resolution, but 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 brivaracetam intermediates using enzymatic asymmetric hydrogenation have been reported. CN107604018A discloses a method for synthesizing (R)-4-propyldihydrofuran-2-one from a substrate 4-n-propyl-2(5H)-furanone catalyzed by ene reductase, but no information such as the ene reductase sequence is disclosed. Feng Jiacheng and Sun Lili et al. also reported a method for preparing (R)-4-propyldihydrofuran-2-one using an 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. However, these reports all have disadvantages such as low substrate concentration, complex operation, and low conversion rate, making them unsuitable 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 alkene 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 alkene 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 alkene reductase mutant; preferably, its nucleotide sequence is shown as 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 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.

[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 as shown in SEQ ID NOs. 4 to 6, respectively.

[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 a 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 for catalyzing 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 complex 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 a phosphate buffer system; 2) nicotinamide adenine dinucleoside phosphate, isopropanol, and a phosphate buffer system; and 3) nicotinamide adenine dinucleoside phosphate, ammonium formate, and a phosphate buffer system.

[0026] Diastereoisomer crystallization involves first reacting a chiral compound with a racemate to form diastereomers, then exploiting the solubility differences between the diastereomers to remove the unwanted diastereomers via crystallization to achieve resolution. Preferably, the diastereomers are obtained by reacting 4-propyldihydrofuran-2(3H)-one with (S)-phenylethylamine, and then recrystallizing to remove the isomers.

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

[0028] The present invention provides a composite enzyme composed of a mutant M8 of olefin reductase E1, a carbonyl reductase K1, and a glucose dehydrogenase. The composite enzyme 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 through diastereoisomer crystallization. The composite enzyme can be used for the one-pot biocatalytic preparation of key intermediates of brivaracetam, thereby reducing production costs, reducing the use of polluting chemicals, and reducing 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 This is the chiral liquid chromatography 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] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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 the pET22b(+) vector, respectively. The clones were then introduced into the host Escherichia coli BL21(DE3) competent cells and cultured on ampicillin-resistant plates. Single colonies were picked and cultured in LB medium, and finally recombinant genetically engineered bacteria expressing ene reductases E1, E2, E3, E4, E5, E6, E7, and E8 were obtained, respectively.

[0038] Recombinant genetically engineered bacteria E1, E2, E3, E4, E5, E6, E7, and E8 were selected and added to 5 mL of LB medium (containing 50 μg / mL ampicillin) and cultured overnight at 37°C, 220 rpm to obtain a seed solution. 10 μL of the seed solution was transferred to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C, 220 rpm. When the OD600 value was >0.8, isopropylthiogalactoside (IPTG) was added to a final concentration of 0.2 mM to induce expression of ene reductases E1, E2, E3, E4, E5, E6, E7, and E8 at 25°C and cultured overnight. After fermentation, the cells were collected by centrifugation at 12,000 g for 10 minutes.

[0039] Table 1 Olefin reductases 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 cloned into the pET22b(+) vector. The cells were then introduced into competent Escherichia coli BL21(DE3) cells and cultured on ampicillin-resistant plates. Single colonies were picked and cultured in LB medium to obtain recombinant glucose dehydrogenase-encoding bacteria. Fermentation was performed using the same method as in Example 1 above and the cells were then used.

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

[0045]

[0046] 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 a 50% aqueous solution of glyoxylic acid dropwise, controlling the rate of addition to maintain the temperature below 32°C. After completion of the addition, react at room temperature for 1 hour. Cool in an ice bath and add 125 g of valeraldehyde dropwise, controlling the internal temperature below 40°C during the addition. After completion of the addition, react at 40°C for 20 hours, then cool to room temperature. Add 240 g of concentrated hydrochloric acid dropwise to adjust the pH of the aqueous layer to below 5. Separate the liquid, wash the aqueous phase with heptane (500 mL x 2), extract with isopropyl ether (1000 mL x 3), separate the liquid, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 134 g 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, and 2 mL phosphate buffer (pH 7.0). 50 g / L of ene reductases E1, E2, E3, E4, E5, E6, E7, and E8 were then added to the reaction solution. After 6 hours of reaction at 220 rpm and 30°C, the conversion of substrate 4 to 5 and the chirality of product 1 were determined, as shown in Table 2. E1 exhibited the best activity and chirality, with a conversion of 75.2% for substrate 4 to 5 and 9.1% for 4 to 1, resulting in 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: maintain at 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 in methanol and dilute it to a 10mg / mL solution.

[0053] Chiral liquid phase detection method for enzyme-catalyzed products: chromatographic column: Phenylephrine Chiral NX(2) 5μ; mobile phase: 90% n-hexane and 10% isopropanol; column temperature: 30°C; detection wavelength: 210 nm; flow rate: 0.5 mL / 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 cloned into the pET22b(+) vector. The clones were then introduced into the competent host Escherichia coli BL21(DE3) cells and cultured on ampicillin-resistant plates. Single colonies were picked and cultured in LB medium to obtain recombinant genetically engineered bacteria expressing carbonyl reductases K1, K2, K3, K4, and K5, respectively.

[0057] Pick the glycerol strains of the recombinant genetically engineered bacteria K1, K2, K3, K4, and K5 mentioned above and add them to 5 mL of LB medium (containing 50 μg / mL ampicillin) and culture them overnight at 37°C and 220 rpm to obtain seed liquid. Pipette 10 μL of seed liquid and transfer it to a shake flask containing 50 mL of fermentation medium (containing 50 μg / mL ampicillin) and culture it at 37°C and 220 rpm. When the OD600 value is >0.8, add isopropylthiogalactoside (IPTG) at a final concentration of 0.2 mM 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 10 minutes 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 + , 20 g / L glucose, 50 g / L ene reductase E1, 25 g / L glucose dehydrogenase, and 2 mL phosphate buffer (pH 7.0). 50 g / L carbonyl reductases K1, K2, K3, K4, and K5 were then added to the reaction solution. After reacting at 220 rpm and 30°C for 6 hours, the conversion rate and product chirality were measured. The results are shown in Table 3. The combination of ene reductase E1 and carbonyl reductase K1 achieved the best conversion rate, with a substrate conversion rate of 100% and a product ee value of 39.3%.

[0060] 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

[0061]

[0062]

[0063] Example 6 Construction and Screening of an 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 from the substrate. Site-directed and combinatorial saturation mutagenesis were performed on a range of amino acids. The constructed mutant plasmid library was transformed into Escherichia coli BL21(DE3), plated onto LB solid medium containing 50 μg / mL ampicillin, and cultured overnight in a 37°C incubator. Single colonies were picked and transferred to a 96-well plate containing 400 μL LB medium (containing 50 μg / mL ampicillin) and cultured overnight at 37°C, 200 rpm to obtain a 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. Isopropylthiogalactoside (IPTG) was used to induce the expression of the mutant at 28°C at a final concentration of 1 mM, 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 bacteria. The cells were then lysed with 200 μL of lysis buffer (0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.5). 7.0) resuspended the cells, then lysed at 30°C for 1 hour, centrifuged at 4°C, 4000g, and centrifuged for 30 minutes. The clarified supernatant was aspirated to determine the activity of the mutant. 190 μL of the reaction solution (containing 0.5 m / min) was used to detect changes in NADPH at 340 nm. The NADPH consumption was used to calculate the enzyme activity of the reaction mutant. A portion of the mutant was selected and added to a 2 mL reaction for re-testing. After the mutant strain was fermented according to Example 1, carbonyl reductase K1 was added to the reaction. 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.

[0065] Table 4. Ene reductase 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 an activity-enhancing 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 0.5g of mutant M8 of ene reductase E1, 0.1g of 5-hydroxy-4-n-propyl-2-furanone, and 1mg of NADP into the reaction bottle. +0.5 g carbonyl reductase K1, 0.5 g glucose, 0.25 g glucose dehydrogenase, and 10 mL phosphate buffer (pH 7.0) were shaken for 8 h at 220 rpm, with the pH controlled at 7.0-8.0. The substrate conversion was 100% and the product ee was 93.0%.

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

[0072] One-pot preparation: Add 45g of E1 mutant M8 bacteria, 45g of carbonyl reductase K1 bacteria, 27g of glucose dehydrogenase bacteria, 0.54g of 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 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 to obtain 73.9g of yellow oil, molar yield 91.1%, purity about 96%, ee 93.0%, gas phase spectrum and chiral liquid phase spectrum are as follows 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 resulting solid is removed by concentrated sulfuric acid to remove (S)-phenylethylamine and ring closure (a chain structure is formed after removal of phenylethylamine, and acid needs to be added to close the ring to obtain a five-membered ring lactone structure) to obtain the brivaracetam 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, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. An alkene 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 alkene 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 complex 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 alkene 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 alkene 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 complex 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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