Ketene reductase and method for preparing (R)-4-propyl-dihydrofuran-2-ketone by using ketene reductase

Abstract

The invention discloses ketene reductase and a method for preparing (R)-4-propyl-dihydrofuran-2-ketone by using the ketene reductase. Compared with an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence of the ketene reductase has at least 95%, 96%, 97%, 98% or 99% of identity; and at least any one or more of differences of amino acid residues I67, C25 and Y27 and homologous sites thereof exist. According to the method, 5-hydroxy-4-n-propyl-2 (5H)-furanone is used as a substrate, the ketene reductase is used for enzyme catalysis to prepare the product (R)-4-propyl-dihydrofuran-2-ketone, the substrate is wide in source and low in price, hydroxyl does not need to be removed in advance in the catalytic reaction, one-pot reaction can be achieved, the total reaction time is short, and the synthesis efficiency of the product is high.

Description

Technical Field

[0001] This invention belongs to the field of biological enzyme catalysis, specifically relating to an enone reductase and a method for preparing (R)-4-propyl-dihydrofuran-2-one using it. Background Technology

[0002] Brivaracetam is a novel antiepileptic drug developed by UCB Pharma in Belgium. It has a high affinity for synaptic vesicle protein 2A (SV2A) and is used as adjunctive therapy for partial seizures, with or without secondary generalized seizures, in adults and adolescents aged 16 years and older with epilepsy. The intermediate of brivaracetam is (R)-4-propyl-dihydrofuran-2-one, with the structural formula shown in Formula 1 below.

[0003]

[0004] (R)-4-propyl-dihydrofuran-2-one can be prepared by chemical methods. For example, WO2016191435 uses (R)-epoxychloropropane as a starting material to prepare (R)-4-propyl-dihydrofuran-2-one through a three-step reaction, and then uses (R)-4-propyl-dihydrofuran-2-one as an intermediate to further prepare buvasitan. The reaction process is shown in Equation 2 below.

[0005]

[0006] For example, CN106588741A uses (R)-3-methoxycarbonylhexanoic acid as a starting material, which is cyclized to obtain (R)-4-propyl-dihydrofuran-2-one. Then, (R)-4-propyl-dihydrofuran-2-one is further subjected to ring-opening, substitution, and cyclization to obtain buvasitan. The reaction process is shown in Equation 3 below.

[0007]

[0008] The preparation of (R)-4-propyl-dihydrofuran-2-one by chemical methods requires the use of chiral raw materials, which is costly.

[0009] Alternatively, (R)-4-propyl-dihydrofuran-2-one can also be prepared using enzymatic catalysis. For example, CN111154735B provides a method for preparing (R)-4-propyl-dihydrofuran-2-one by using an enone reductase derived from Candida castelii to catalyze the reduction reaction of 4-n-propyl-2(5H)-furanone. The reaction conditions are: reaction at 30°C for 24 h, with an ee value of 99.4% for the target product. However, this method has a long reaction time, which is not conducive to industrial production.

[0010] For example, CN113444702B discloses a mutant OYE296A of the enone reductase OYE1 (NCBI accession number: Q02899) from Saccharomyces pastorianus, which can catalyze the asymmetric reduction of 4-propyl-2(5H)-furanone with a substrate conversion rate greater than 99% and an ee value of the product greater than 99%. However, this method requires a reaction time of more than 12 hours, which is not conducive to industrial production. Summary of the Invention

[0011] The technical problem to be solved by the present invention is that the cost of preparing (R)-4-propyl-dihydrofuran-2-one by existing chemical methods is high and the reaction time of existing enzyme catalysis methods is long, which is not conducive to industrial production. The present invention provides an enone reductase and a method for preparing the buvastan intermediate (R)-4-propyl-dihydrofuran-2-one using it.

[0012] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0013] A first aspect of the present invention provides an enone reductase having an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:1, and having at least one or more differences among amino acid residues I67, C25, Y27 and their homologous sites, wherein the positions of 67, 25, and 27 refer to the amino acid residue numbers in SEQ ID NO:1.

[0014] In this invention, homologous sites refer to the corresponding sites between amino acid sequences that have a certain degree of identity, determined by combining the results of protein three-dimensional structure analysis during amino acid sequence alignment.

[0015] In some embodiments of the present invention, the amino acid residues of I67 are different from those of I67A, I67C, I67D, I67E, I67F, I67G, I67H, I67K, I67L, I67M, I67N, I67P, I67Q, I67R, I67S, I67T, I67V, I67W or I67Y.

[0016] In some embodiments of the present invention, the amino acid residues of C25 are different from those of C25A, C25D, C25E, C25F, C25G, C25H, C25I, C25K, C25N, C25P, C25Q, C25R, C25S, C25T, C25W or C25Y.

[0017] In some embodiments of the present invention, the amino acid residues of Y27 are different from those of Y27A, Y27C, Y27D, Y27E, Y27F, Y27H, Y27I, Y27K, Y27N, Y27P, Y27Q, Y27R, Y27S, Y27V or Y27W.

[0018] In some embodiments of the present invention, the amino acid sequence of the enone reductase has the following amino acid residues compared to the amino acid sequence shown in SEQ ID NO:1: I67A, I67C, I67D, I67E, I67F, I67G, I67H, I67K, I67L, I67M, I67N, I67P, I67Q, I67R, I67S, I67T, I67V, I67W, I67Y, C25A, C25D, C25E, C25F, C25G, C25H, C25I, C25K, C25N, C25 Differences between P, C25Q, C25R, C25S, C25T, C25W, C25Y, Y27A, Y27C, Y27D, Y27E, Y27F, Y27H, Y27I, Y27K, Y27N, Y27P, Y27Q, Y27R, Y27V, Y27W, C25A / I67A, C25A / I67V, C25G / I67A, C25G / I67V, C25S / I67A, or C25S / I67V.

[0019] In this invention, " / " indicates that the contents described before and after it coexist. For example, C25A / I67A indicates that the amino acid residues at positions 25 and 67 in the amino acid sequence are different at the same time.

[0020] In some preferred embodiments of the present invention, the amino acid sequence of the enone reductase differs from the amino acid sequence shown in SEQ ID NO:1 in the following amino acid residues: I67A, I67V, I67C, I67D, I67F, I67G, I67H, I67L, I67N, I67P, I67R, I67S, I67T, I67Y, C25A, C25G, C25K, C25N, C25S, C25Y, C25A / I67A, C25A / I67V, C25G / I67A, C25G / I67V, C25S / I67A, or C25S / I67V.

[0021] A second aspect of the present invention provides a multi-enzyme system comprising an enone reductase and a ketone reductase; the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase described in the first aspect of the present invention; the amino acid sequence of the ketone reductase is as shown in SEQ ID NO:7.

[0022] In some embodiments of the present invention, the multi-enzyme system further comprises dehydrogenases, wherein the dehydrogenases are formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase.

[0023] In some specific embodiments of the present invention, the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO:9.

[0024] In some specific embodiments of the present invention, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:11.

[0025] In some preferred embodiments of the present invention, the various enzyme components in the multi-enzyme system may exist individually or together in the form of whole-cell enzymes, homogenized enzyme solutions, crude enzyme solutions, purified enzyme solutions, solid enzyme powders, or immobilized enzymes.

[0026] In this invention, whole-cell enzymes are produced by cells (e.g., prokaryotic cells) containing genes encoding any one or more enzymes in a multi-enzyme system; whole-cell catalysis refers to the catalysis of substrates using a complete biological organism (i.e., a cell, tissue, or even an individual), which is essentially the catalysis using enzymes within the cell.

[0027] In this invention, the homogenized enzyme solution is an enzyme solution obtained by resuspending wet bacterial cells in a resuspension solution and then disrupting the cells. The wet bacterial cells are the precipitate obtained by solid-liquid separation (i.e., centrifugation followed by discarding the supernatant and collecting the precipitate) of the prokaryotic cell culture medium.

[0028] In this invention, the methods for cell disruption include, but are not limited to: high-pressure disruption, ultrasonic disruption, osmotic shock disruption, repeated freeze-thaw cycles, lysozyme treatment, or cell lysis buffer treatment.

[0029] In this invention, the crude enzyme solution is the enzyme solution obtained by removing impurities from the homogenized enzyme solution (i.e., centrifuging, taking the supernatant, and discarding the precipitate).

[0030] In this invention, the purified enzyme solution is the enzyme solution obtained by purifying the crude enzyme solution (i.e. removing impurities and proteins).

[0031] In this invention, solid enzyme powder is a powder obtained by drying and pulverizing crude enzyme solution or purified enzyme solution.

[0032] In this invention, the immobilized enzyme is an enzyme in an immobilized form obtained by confining a free enzyme within a certain space or attaching it to a solid structure.

[0033] A third aspect of the present invention provides an isolated nucleic acid or a nucleic acid composition, wherein the nucleic acid encodes an enone reductase as described in the first aspect of the present invention; the nucleic acid composition comprises a nucleic acid encoding an enzyme in a multi-enzyme system as described in the second aspect of the present invention.

[0034] In some embodiments of the present invention, the nucleotide differences corresponding to the amino acid residue differences of the enone reductase are as follows: ATT67GCT, ATT67TGT, ATT67GAT, ATT67GAG, ATT67TTT, ATT67GGT, ATT67CAT, ATT67AAG, ATT67CTG, ATT67ATG, ATT67AAT, ATT67CCG, ATT67CAA, ATT67CGT, ATT67AGT, ATT67ACT, ATT67GTT, ATT67TGG, ATT67TAT, TGC25GCG, TGC25GAT, TGC25GAA, TGC25TT T, TGC25GGA, TGC25CAT, TGC25ATT, TGC25AAG, TGC25AAT, TGC25CCG, TGC25CAA, TGC25AGA, TGC25AGC, TGC25ACG, TGC25TGG, TGC25TAT, TAT27GCG, TAT 27TGT, TAT27GAT, TAT27GAG, TAT27TTT, TAT27CAT, TAT27ATT, TAT27AAG, TAT27AAT, TAT27CCT, TAT27CAG, TAT27CGT, TAT27AGT, TAT27GTG and TAT27TGG.

[0035] In this invention, the numbers represent the encoded amino acid sites, and the three nucleotides before and after the numbers correspond to the nucleotide sequences before and after the mutation, respectively. For example, "ATT67GCT" indicates that the nucleotide sequence corresponding to the 67th amino acid is mutated from ATT to GCT.

[0036] A fourth aspect of the present invention provides a recombinant expression vector comprising a nucleic acid or nucleic acid composition as described in the third aspect of the present invention.

[0037] In some preferred embodiments of the present invention, the recombinant expression vector comprises a pET28a plasmid backbone.

[0038] A fifth aspect of the present invention provides a transformant comprising: a host cell; and, introduced into the host cell, an isolated nucleic acid or nucleic acid composition as described in a third aspect of the present invention, or a recombinant expression vector as described in a fourth aspect of the present invention.

[0039] In some embodiments of the present invention, the host cell includes eukaryotic cells and prokaryotic cells.

[0040] In some embodiments of the present invention, the host cell is a bacterium.

[0041] In some embodiments of the present invention, the bacteria are Escherichia coli.

[0042] In some specific embodiments of the present invention, the bacteria is Escherichia coli BL21(DE3).

[0043] A sixth aspect of the present invention provides a method for preparing intermediate product 1, the method comprising: in the presence of a cofactor, reacting 5-hydroxy-4-n-propyl-2(5H)-furanone with an enone reductase to obtain intermediate product 1;

[0044] The intermediate product 1 is a compound as shown in Formula 1:

[0045]

[0046] In some embodiments of the present invention, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in the first aspect of the present invention.

[0047] In some embodiments of the present invention, the cofactor is NADPH and / or NADH.

[0048] In some embodiments of the present invention, the concentration of the 5-hydroxy-4-n-propyl-2(5H)-furanone added in the reaction is 2-80 mg / mL, preferably 5-60 mg / mL, for example 10 mg / mL or 60 mg / mL.

[0049] In some embodiments of the present invention, when calculating the added mass of the enone reductase according to the mass of the wet bacterial cells that produce the enone reductase, the added mass ratio of the enone reductase to the added mass of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.02-2.5):1, preferably (0.05-2.2):1, for example 0.1:1.

[0050] In this invention, the added mass refers to the mass of the component after it has been completely added to the reaction system, before the reaction has started. If the component is added over a period of time, the total mass added to the reaction system when all the component has been added is taken as the added mass. The added volume and added molar amount are treated similarly.

[0051] In some preferred embodiments of the present invention, the cofactor is obtained by the following cofactor regeneration step: reduction of NADP in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + .

[0052] In some preferred embodiments of the present invention, the NADP + and / or NAD + The mass ratio of the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.01-0.5):1, preferably (0.015-0.4):1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1.

[0053] In some preferred embodiments of the present invention, the dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formic acid or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol.

[0054] When the hydrogen donor is formic acid or formate, the dehydrogenase is formate dehydrogenase; when the hydrogen donor is D-glucose-6-phosphate, the dehydrogenase is glucose-6-phosphate dehydrogenase; when the hydrogen donor is C1-C3 fatty alcohol, the dehydrogenase is alcohol dehydrogenase; when the hydrogen donor is glucose, the dehydrogenase is glucose dehydrogenase.

[0055] In some preferred embodiments of the present invention, when calculating the added mass of the dehydrogenase according to the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1.

[0056] In some specific embodiments of the present invention, when the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%.

[0057] In some specific embodiments of the present invention, when the hydrogen donor is glucose, the molar ratio of the added glucose to the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (1-5):1, preferably (1-3):1, for example 2.7:1 or 2.1:1.

[0058] A seventh aspect of the present invention provides a method for preparing intermediate product 2, the method comprising: reacting intermediate product 1 with a ketone reductase in the presence of a cofactor to obtain intermediate product 2;

[0059] The intermediate product 1 is a compound as shown in Formula 1:

[0060]

[0061] The intermediate product 2 is a compound as shown in Formula 2:

[0062]

[0063] In some embodiments of the present invention, the amino acid sequence of the ketone reductase is shown in SEQ ID NO:7.

[0064] In some embodiments of the present invention, the cofactor is NADPH and / or NADH.

[0065] In some embodiments of the present invention, the concentration of intermediate product 1 added to the reaction system is 2-80 mg / mL, preferably 5-60 mg / mL, for example 10 mg / mL or 60 mg / mL.

[0066] In some embodiments of the present invention, when calculating the added mass of the ketone reductase according to the mass of the wet bacterial cells that produce the ketone reductase, the ratio of the added mass of the ketone reductase to the added mass of the intermediate product 1 is (0.05-10):1, preferably (0.1-5):1, for example 2:1 or 0.1:1.

[0067] In some preferred embodiments of the present invention, the cofactor is obtained by the following cofactor regeneration step: reduction of NADP in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + .

[0068] In some preferred embodiments of the present invention, the NADP + and / or NAD + The mass ratio of the intermediate product 1 to the added intermediate product is (0.01-0.5):1, preferably (0.015-0.4):1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1.

[0069] In some preferred embodiments of the present invention, the dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formic acid or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol.

[0070] In some preferred embodiments of the present invention, when calculating the added mass of the dehydrogenase according to the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to the added mass of the intermediate product 1 is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1.

[0071] In some specific embodiments of the present invention, when the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%.

[0072] In some specific embodiments of the present invention, when the hydrogen donor is glucose, the molar ratio of glucose to intermediate product 1 is (1-5):1, preferably (1-3):1, for example 2.7:1 or 2.1:1.

[0073] In some embodiments of the present invention, the intermediate product 1 is prepared by the method as described in the sixth aspect of the present invention.

[0074] An eighth aspect of the present invention provides a method for preparing the buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one, the method comprising:

[0075] Intermediate product 1 was obtained by catalyzing the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone with enone reductase, and (R)-4-propyl-dihydrofuran-2-one was prepared from said intermediate product 1.

[0076] The intermediate product 1 is a compound as shown in Formula 1:

[0077]

[0078] In some embodiments of the present invention, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in the first aspect of the present invention.

[0079] In some embodiments of the present invention, the method for obtaining intermediate 1 by catalyzing the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone with enone reductase is as described in the sixth aspect of the present invention.

[0080] In some embodiments of the present invention, the preparation of (R)-4-propyl-dihydrofuran-2-one using the intermediate product 1 specifically includes: using ketone reductase to catalyze the reaction of intermediate product 1 to obtain intermediate product 2; intermediate product 2 undergoes a ring-closing reaction and is finally converted into the (R)-4-propyl-dihydrofuran-2-one.

[0081] The intermediate product 2 is a compound as shown in Formula 2:

[0082]

[0083] In some preferred embodiments of the present invention, the amino acid sequence of the ketone reductase is shown in SEQ ID NO:7.

[0084] In some preferred embodiments of the present invention, the method of using ketone reductase to catalyze the reaction of intermediate 1 to obtain intermediate 2 is as described in the seventh aspect of the present invention.

[0085] A ninth aspect of the present invention provides a method for preparing the buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one, the method comprising:

[0086] Intermediate product 1 was reacted with ketone reductase to obtain intermediate product 2; intermediate product 2 was then converted into (R)-4-propyl-dihydrofuran-2-one via a ring-closure reaction.

[0087] The intermediate product 1 is a compound as shown in Formula 1:

[0088]

[0089] The intermediate product 2 is a compound as shown in Formula 2:

[0090]

[0091] In some embodiments of the present invention, the amino acid sequence of the ketone reductase is shown in SEQ ID NO:7.

[0092] In some embodiments of the present invention, the method of using ketone reductase to catalyze the reaction of intermediate 1 to obtain intermediate 2 is as described in the seventh aspect of the present invention.

[0093] In some embodiments of the present invention, the method further includes: using enone reductase to catalyze the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone to obtain the intermediate product 1.

[0094] In some preferred embodiments of the present invention, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in the first aspect of the present invention.

[0095] In some preferred embodiments of the present invention, the method for obtaining intermediate 1 by catalyzing the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone with enone reductase is as described in the sixth aspect of the present invention.

[0096] The tenth aspect of the present invention provides a method for preparing the buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one, the method comprising: reacting 5-hydroxy-4-n-propyl-2(5H)-furanone in the presence of a cofactor under the catalysis of an enone reductase and a ketone reductase to generate intermediate 2, followed by a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one;

[0097] The intermediate product 2 is a compound as shown in Formula 2:

[0098]

[0099] In some embodiments of the present invention, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in the first aspect of the present invention.

[0100] In some embodiments of the present invention, the amino acid sequence of the ketone reductase is shown in SEQ ID NO:7.

[0101] In some embodiments of the present invention, the 5-hydroxy-4-n-propyl-2(5H)-furanone is added by adding it to a mixture containing the enone reductase and the ketone reductase over a period of time.

[0102] In some embodiments of the present invention, the ratio of the added mass of 5-hydroxy-4-n-propyl-2(5H)-furanone to the final total volume of the reaction system is 2-80 mg:1 mL, preferably 5-60 mg:1 mL, for example 10 mg:1 mL or 60 mg:1 mL.

[0103] In some embodiments of the present invention, when calculating the added mass of the enone reductase according to the mass of the wet bacterial cells that produce the enone reductase, the added mass ratio of the enone reductase to the added mass of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.02-2.5):1, preferably (0.05-2.2):1, for example 0.1:1.

[0104] In some embodiments of the present invention, when calculating the added mass of the ketone reductase according to the mass of the wet bacterial cells that produce the ketone reductase, the ratio of the added mass of the ketone reductase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-10):1, preferably (0.1-5):1, for example 2:1 or 0.1:1.

[0105] In some embodiments of the present invention, the cofactor is NADPH and / or NADH.

[0106] In some preferred embodiments of the present invention, the addition is carried out at a constant rate.

[0107] In some preferred embodiments of the present invention, the NADPH and / or NADH are obtained by the following cofactor regeneration step: reduction of NADP in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + .

[0108] In some preferred embodiments of the present invention, the NADP + and / or NAD + The mass ratio of the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.01-0.5):1, preferably (0.015-0.4):1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1.

[0109] In some preferred embodiments of the present invention, the addition rate of the 5-hydroxy-4-n-propyl-2(5H)-furanone is 5-9 g / h, for example 7.5 g / h.

[0110] In some preferred embodiments of the present invention, the dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formic acid or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol.

[0111] In some preferred embodiments of the present invention, when calculating the added mass of the dehydrogenase according to the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1.

[0112] In some specific embodiments of the present invention, when the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%.

[0113] In some specific embodiments of the present invention, when the hydrogen donor is glucose, the molar ratio of the added glucose to the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (2-6):1, preferably (2-4):1, for example 2.7:1 or 2.1:1.

[0114] In some embodiments of the present invention, the pH value of the reaction is 5-8.

[0115] In some embodiments of the present invention, the reaction temperature is 20-35°C.

[0116] In some embodiments of the present invention, the reaction time is 0.5-21 h.

[0117] In some embodiments of the present invention, the ring-closing reaction is carried out under acidic and heated conditions.

[0118] In some preferred embodiments of the present invention, the pH value is adjusted by a buffer solution or an acid-base solution, wherein the buffer solution is preferably a PBS buffer solution, and the acid-base solution is preferably a hydrochloric acid solution or a sodium hydroxide solution.

[0119] In some preferred embodiments of the invention, the pH value of the acidic conditions is <2.0.

[0120] In some preferred embodiments of the present invention, the heating conditions are a water bath at 60-80°C, for example, a water bath at 70°C.

[0121] In some preferred embodiments of the present invention, the duration of the heating conditions is 20-60 minutes, for example, 30 minutes.

[0122] The eleventh aspect of the present invention provides an enzyme reaction system comprising an enone reductase or a multi-enzyme system, and 5-hydroxy-4-n-propyl-2(5H)-furanone;

[0123] The amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in the first aspect of the present invention; the multi-enzyme system is the multi-enzyme system as described in the second aspect of the present invention.

[0124] In some embodiments of the present invention, the enzyme reaction system catalyzes the reaction as described in the sixth, eighth, ninth, or tenth aspects of the present invention.

[0125] The twelfth aspect of the present invention provides an enzyme reaction system comprising a ketone reductase and intermediate product 1;

[0126] The intermediate product 1 is a compound as shown in Formula 1:

[0127]

[0128] The amino acid sequence of the ketone reductase is shown in SEQ ID NO:7.

[0129] In some embodiments of the present invention, the enzyme reaction system catalyzes the reaction as described in the seventh, eighth, or ninth aspects of the present invention.

[0130] The thirteenth aspect of the present invention provides the use of an amino acid reductase as shown in SEQ ID NO:1 or having a difference of amino acid residue Y27S compared to the amino acid sequence shown in SEQ ID NO:1, or as described in the first aspect of the present invention; a ketone reductase as shown in SEQ ID NO:7; a multi-enzyme system as described in the second aspect of the present invention; a nucleic acid or nucleic acid composition as described in the third aspect of the present invention; a recombinant expression vector as described in the fourth aspect of the present invention; a transformant as described in the fifth aspect of the present invention; or an enzyme reaction system as described in the eleventh or twelfth aspect of the present invention in the preparation of buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one or buvasidan using 5-hydroxy-4-n-propyl-2(5H)-furanone or intermediate 1;

[0131] The structure of intermediate product 1 is shown in Formula 1:

[0132]

[0133] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0134] The reagents and raw materials used in this invention are all commercially available.

[0135] The positive and progressive effects of this invention include any one or more of the following:

[0136] This invention uses 5-hydroxy-4-n-propyl-2(5H)-furanone as a substrate and employs an enzymatic catalytic method to prepare the product (R)-4-propyl-dihydrofuran-2-one. This substrate is widely available and inexpensive, and the reaction conditions of the enzymatic catalysis are relatively mild, resulting in low overall preparation costs. Furthermore, the substrate does not require prior removal of its hydroxyl group during the enzymatic reaction; therefore, the enzymatic catalytic reaction system of this invention does not require the addition of a dehydroxylation reagent, which simplifies the reaction route and facilitates a one-pot reaction.

[0137] This invention utilizes a one-pot reaction system to prepare the product from the substrate. This one-pot reaction system simultaneously contains both enone reductase and ketone reductase, enabling direct synthesis of the product without the need to separate and purify intermediate 1 or prepare the reaction system multiple times. This reduces the loss of intermediate 1 and simplifies the operation. Furthermore, the ketone reductase catalyzes the conversion of intermediate 1, which helps to accelerate the reaction process of enone reductase and improve the substrate conversion rate.

[0138] In this invention, the substrate can be added to the reaction system at a uniform rate over a period of time. This allows the substrate to be rapidly converted by the enone reductase, thereby avoiding excessively high substrate concentrations that could affect enzyme activity and increasing the total amount of substrate added.

[0139] The one-pot reaction of the present invention has a total reaction time of only 3.5 to 6.5 hours, which is relatively short. The synthesis efficiency of the final product is high, and the ee value of the final product can reach more than 92%, which is beneficial to industrial production. Attached Figure Description

[0140] Figure 1 This is a schematic diagram of the reaction route for preparing the final product (R)-4-propyl-dihydrofuran-2-one using 5-hydroxy-4-n-propyl-2(5H)-furanone as a substrate. In this diagram, OYE (old yellow enzyme) represents old yellow enzyme, also known as ketone reductase in this invention; KRED represents ketoreductase; ADH represents isopropanol oldehydrogenase; IPA represents isopropanol; GDH represents glucose dehydrogenase; Glc represents glucose; and GluAc represents gluconic acid. ADH / GDH represents alcohol dehydrogenase or glucose dehydrogenase. IPA / Glc represents isopropanol or glucose. Acetone / GluAc represents acetone or gluconic acid. Detailed Implementation

[0141] The present invention is further illustrated below by way of examples, but the invention is not limited to the scope of the examples described herein. Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available.

[0142] TB liquid culture medium: peptone 10 g / L, yeast extract 18 g / L, glycerol 0.4% (v / v), KH2PO4 (anhydrous) 2.31 g / L, K2HPO4 (trihydrate) 16.43 g / L.

[0143] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0144] LB agar plates: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 18g / L.

[0145] The substrate 5-hydroxy-4-n-propyl-2(5H)-furanone was synthesized according to WO2017076738A1. The reference standard (R configuration) of the final product (R)-4-propyl-dihydrofuran-2-one was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., catalog number BD626939-1g. The reference standard of the racemic form of the final product (containing both R and S configurations) was synthesized according to CN109134406A.

[0146] BugBuster protein extraction reagent ( Protein Extraction Reagent was purchased from Merck Millipore, product number 70584-4.

[0147] The gas chromatography method for detecting the ee value of the final product in this embodiment is as follows:

[0148] Chromatographic conditions: Column: LIPODEX E, 25m × 0.25mm × 1μm; Flow rate: 0.7mL / min; Split ratio: 30:1; Injection volume: 0.5μL; Injector temperature: 220℃; Detector temperature: 240℃; Column oven temperature: Initial temperature 130℃, hold for 16min, increase to 180℃ at a rate of 20℃ / min, hold for 10min; Hydrogen: 30mL / min; Air: 300mL / min; Make-up purge: 25mL / min.

[0149] Reference retention times: The retention times of the racemic final product reference were 17.660 min (R configuration) and 18.329 min (S configuration), and the retention time of the final product (R)-4-propyl-dihydrofuran-2-one was 17.569 min.

[0150] Formula for calculating the ee value of the final product: Among them, AR : Peak area of ​​the final product with R configuration, A S : Peak area of ​​the S-configuration final product.

[0151] The gas chromatography method for detecting the conversion rate of the substrate in the examples is as follows:

[0152] Chromatographic conditions: Column: Agilent HP-5, 30m × 0.32mm × 0.25μm; Column flow rate: 1.0mL / min; Split ratio: 25:1; Injection volume: 0.5μL; Injector temperature: 250℃; Detector temperature: 280℃; Oven temperature: Initial temperature 40℃, hold for 2 min, then increase to 200℃ at a rate of 20℃ / min, hold for 3 min, then increase to 270℃ at a rate of 40℃ / min, hold for 5 min; Hydrogen: 30mL / min; Air: 300mL / min; Make-up purge: 25mL / min.

[0153] Reference retention times: substrate 9.842 min, intermediate 1 9.040 min, final product 8.419 min.

[0154] Formula for calculating substrate conversion rate: Among them, A 底 A1: Peak area of ​​the substrate; A2: Sum of peak areas of the substrate, intermediate product 1, and final product.

[0155] The enzyme catalytic reaction route of the present invention is as follows: Figure 1 As shown in the figure, the substrate undergoes ring-opening in the enzyme-catalyzed reaction system. The enone reductase catalyzes the ring-opening substrate to convert it into intermediate product 1. Then, the ketone reductase catalyzes the conversion of intermediate product 1 into intermediate product 2. Subsequently, intermediate product 2 undergoes ring-closure under acidic and heated conditions, and is finally converted into the final product (R)-4-propyl-dihydrofuran-2-one.

[0156] Furthermore, ketone reductases and enone reductases require reducing cofactors NADPH or NADH to continuously provide hydrogen for their respective enzymatic catalytic reactions. To achieve the cyclic regeneration of reducing cofactors NADPH or NADH and thus reduce the amount of cofactors added, this invention introduces alcohol dehydrogenase or glucose dehydrogenase into the enzyme catalytic reaction system. Alcohol dehydrogenase catalyzes the conversion of isopropanol to acetone, while simultaneously converting NADP generated in the enzyme catalytic reaction system... + or NAD + They are reduced to NADPH or NADH, respectively. Glucose dehydrogenase catalyzes the conversion of glucose to gluconic acid, while simultaneously reducing NADP produced in the enzyme-catalyzed reaction system. + or NAD + They are reduced to NADPH or NADH respectively.

[0157] Example 1: Acquisition of enzyme gene and preparation of crude enzyme solution

[0158] 1.1 Synthesis of enzyme genes

[0159] To construct an enzymatic reaction system catalyzing the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone to (R)-4-propyl-dihydrofuran-2-one, the gene sequences of enone reductases E1-E3, ketone reductase E4, alcohol dehydrogenase E5, and glucose dehydrogenase E6 were retrieved from the NCBI database. The obtained gene sequences of enone reductases and ketone reductases, as well as the gene sequences of alcohol dehydrogenases and glucose dehydrogenases used for cofactor regeneration, underwent codon optimization and were synthesized at Sangon Biotech (Shanghai) Co., Ltd. These sequences were then cloned into the expression vector pET28a, yielding multiple recombinant plasmids. Each recombinant plasmid contains the encoding gene for only one enzyme. The sources and sequences of all enzymes are shown in Table 1, and all use NADPH as a cofactor.

[0160] Table 1. Sources and sequence numbers of different enzymes

[0161]

[0162] 1.2 Transformation of enzyme genes

[0163] The synthesized recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and successfully transformed recombinant E. coli were screened. The bacterial culture of recombinant E. coli was then plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C until single colonies of recombinant E. coli formed on the LB agar plates.

[0164] 1.3 Expression of enzyme genes

[0165] Single colonies of recombinant *E. coli* from LB plates were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 4 h. Then, a 1% (v / v) inoculation was added to TB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 250 rpm until the bacterial concentration reached OD500. 600 =0.6~0.8, add IPTG to a final concentration of 0.1mM to induce enzyme expression, incubate overnight at 25℃ and 250rpm, centrifuge at 4000rpm for 20min, and collect wet cells.

[0166] For each enzyme, 10g of wet bacterial cells expressing that enzyme were taken and resuspended in 50mM pH 7.0 PBS buffer at a ratio of 1g:10mL (i.e., homogenization ratio; the same applies to Examples 2 to 6 below). The cells were then homogenized, centrifuged, and the precipitate was discarded to obtain different crude enzyme solutions, which were stored at -20℃ for later use. For each type of crude enzyme solution, each 1mL of crude enzyme solution (mother liquor) contained the equivalent of 100mg of enzyme from wet bacterial cells.

[0167] Example 2: Comparison of the activities of enone reductases from different sources in catalyzing one-pot reactions

[0168] Multiple enzyme-catalyzed reaction systems (total volume 1 mL) containing enone reductase and ketone reductase from different sources were constructed according to Table 2 below. These systems utilized alcohol dehydrogenase and isopropanol for coenzyme cycling. Additionally, multiple enzyme-catalyzed reaction systems (total volume 1 mL) containing enone reductase and ketone reductase from different sources were constructed according to Table 3 below. These systems utilized glucose dehydrogenase and glucose for coenzyme cycling.

[0169] Table 2. Components of the one-pot enzyme-catalyzed reaction system (alcohol dehydrogenase / isopropanol coenzyme cycle system)

[0170] Component Name Mother liquor concentration Added amount Initial content in the reaction system crude enone reductase solution 100mg / mL 10μL 1mg / mL crude ketone reductase solution 100mg / mL 0.2mL 20mg / mL crude alcohol dehydrogenase solution 100mg / mL 0.06mL 6mg / mL Substrate / 10mg 10mg / mL Isopropyl alcohol (IPA) / 0.06mL 6% (v / v) PBS buffer (pH 7.0) 0.2M 0.25mL 50mM <![CDATA[NADPNa2]]> 0.2M 25μL 5mM Deionized water / To bring the volume up to 1 mL /

[0171] Table 3. Components of the one-pot enzyme-catalyzed reaction system (glucose dehydrogenase / glucose coenzyme cycle system)

[0172] Component Name Mother liquor concentration Added amount Initial content in the reaction system crude enone reductase solution 100mg / mL 10μL 1mg / mL crude ketone reductase solution 100mg / mL 0.2mL 20mg / mL crude glucose dehydrogenase solution 100mg / mL 0.06mL 6mg / mL Substrate / 10mg 10mg / mL Isopropyl alcohol (IPA) / 0.06mL 6% (v / v) glucose / 34.6 mg 192mM PBS buffer (pH 7.0) 0.2M 0.25mL 50mM <![CDATA[NADPNa2]]> 0.2M 25μL 5mM Deionized water / To bring the volume up to 1 mL /

[0173] In Tables 2 and 3, the initial content of each component in the enzyme catalytic reaction system refers to the initial concentration of each component after it is added to the enzyme catalytic reaction system, that is, the concentration of each component before it has reacted, or the concentration when the reaction time is 0h.

[0174] The steps of the enzyme-catalyzed reaction are as follows: Take a reaction vessel and add all components except the crude enzyme solution according to the amounts shown in Table 2 or Table 3. Adjust the pH of the enzyme-catalyzed reaction system to 6.7 before adding the crude enzyme solution, and then add deionized water to make up to 1 mL. Prepare multiple enzyme-catalyzed reaction systems. Place the different enzyme-catalyzed reaction systems at 30℃ and 220 rpm for 15 min, readjust the pH to 6.7, and continue the reaction for 3 h. Then adjust the pH of the reaction solution to <2.0 with HCl, incubate at 70℃ for 30 min, add 1 mL of ethyl acetate for extraction, take the upper organic phase and filter it with an organic filter, and then use gas chromatography to detect the substrate conversion rate and the ee value of the final product. The results are shown in Table 4.

[0175] Table 4. Reaction results of enzyme-catalyzed reaction systems containing enone reductases from different sources.

[0176] Reaction system numbering Enzyme composition of the reaction system substrate conversion rate ee value of the final product Reaction system A1 E1, E4, E5 87.82％ 82.70％ Reaction system A2 E2, E4, E5 63.24％ 78.72％ Reaction system A3 E3, E4, E5 60.56％ 79.26％ Reaction system A4 E1, E4, E6 86.96％ 82.30％ Reaction system A5 E2, E4, E6 62.72％ 78.06％ Reaction system A6 E3, E4, E6 60.04％ 78.73％

[0177] In Table 4, the substrate is 5-hydroxy-4-n-propyl-2(5H)-furanone, and the final product is (R)-4-propyl-dihydrofuran-2-one. A negative ee value indicates that the predominant configuration of the final product is S-type, and a positive ee value indicates that the predominant configuration of the final product is R-type. This representation method is used in the following examples. In the enzyme-catalyzed reaction systems recorded in Table 4, the final product (structural formula as shown)... Figure 1 As shown, all enzymes exhibit the R configuration, but their ee values ​​are all below 90%, which is insufficient for industrial production. Furthermore, the substrate conversion rates of enzyme-catalyzed reaction systems A2 and A5 (containing enone reductase E2) and reaction systems A3 and A6 (containing enone reductase E3) are lower than those of reaction systems A1 and A4 (containing enone reductase E1). Therefore, further modification of the enone reductase is needed to improve substrate conversion and the ee value of the final product.

[0178] Example 3: Saturation Mutation of Key Amino Acids in the Active Pocket Region of Enone Reductase E1

[0179] 3.1 Construction of a saturated mutant enzyme containing key amino acids in the active pocket region of enone reductase E1

[0180] Analysis of the amino acid sequence (SEQ ID NO:1) of wild-type enone reductase E1 identified C25, Y27, and I67 as key amino acid sites in the active pocket region of enone reductase E1. C25 indicates that the 25th amino acid residue from the N-terminus of the enzyme is cysteine, and the others are similarly identified. Primers were designed based on the nucleotide sequence (SEQ ID NO:2) of wild-type enone reductase E1, and saturation mutations were screened at C25, Y27, and I67. The primer sequences are shown in Table 5.

[0181] The saturation mutation screening method includes the following steps: using the pET28a-E1 recombinant plasmid containing the wild-type enone reductase E1 gene as a template, PCR amplification was performed using the primers in Table 5. The PCR product was digested with DpnI restriction enzyme (used to remove unmutated template after point mutation), purified, and then recombined using seamless cloning technology to obtain the recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, and after recovery, it was plated on LB plates containing 50 μg / mL kanamycin and cultured overnight at 37°C. After colony PCR verification and sequencing verification, the mutant enzyme with the target site mutation was obtained. The sequences of the mutant enzymes are shown in Tables 6 to 8, where the numbers represent amino acid sites, such as I67A indicating that the 67th amino acid is mutated from isoleucine to alanine, and ATT67GCT indicating that the nucleotide sequence corresponding to the 67th amino acid is mutated from ATT to GCT. This representation method is used in the following examples.

[0182] Table 5 Primer sequences for single-point saturation mutant of enone reductase E1

[0183]

[0184]

[0185] In Table 5, C25, Y27, and I67 represent the mutated amino acid sites; NNK is the codon of the degenerate primer, which can encode 20 amino acids and one stop codon; F refers to the forward primer and R refers to the reverse primer.

[0186] 3.2 Comparison of catalytic activities of saturated mutant enzymes of enone reductase E1

[0187] Each saturated mutant enzyme of enone reductase E1 was combined with ketone reductase E4 to construct the enzyme-catalyzed reaction system according to Table 2 of Example 2. The final reaction solution of the enzyme-catalyzed reaction system (i.e., the organic phase obtained after extraction with ethyl acetate from the reaction solution of the enzyme-catalyzed reaction system at the end of the reaction) was detected by gas chromatography, and the conversion rate of the substrate and the ee value of the final product were calculated. The results are shown in Tables 6 to 8.

[0188] Table 6. Enzyme sequences and catalytic effects of single-site saturation mutants at C25 position of enone reductase E1.

[0189]

[0190]

[0191] As shown in Table 6, the ee values ​​of the final products catalyzed by the C25A (E1-1), C25G (E1-5), and C25S (E1-15) mutant enzymes (87.10%, 86.35%, and 88.97%, respectively) were significantly higher than those of the wild-type enone reductase E1 (82.70%), and the substrate conversion rate was also improved.

[0192] Table 7. Enzyme sequences and catalytic effects of enone reductase E1 Y27 single-site saturation mutants.

[0193]

[0194] As shown in Table 7, the ee values ​​of the final products catalyzed by the Y27E (E1-23) and Y27W (E1-38) mutant enzymes (81.44% and 80.00%) are close to those of enone reductase E1 (82.70%). However, the ee values ​​of the final products catalyzed by other Y27 mutant enzymes are lower than those of wild-type enone reductase E1, indicating that the single-point mutation at the Y27 site does not have a significant effect on the catalytic ability of enone reductase E1.

[0195] Table 8. Enzyme sequences and catalytic effects of enone reductase E1 with a single-site saturation mutant at position I67.

[0196]

[0197] As shown in Table 8, most I67 mutant enzymes exhibited substrate conversion rates exceeding 93% and final product ee values ​​exceeding 85%. Among them, the I67A (E1-39) and I67V (E1-55) mutant enzymes produced final product ee values ​​of 91.59% and 90.98%, respectively. The I67A (E1-39) and I67V (E1-55) mutant enzymes also demonstrated the best catalytic performance among all saturated mutant enzymes at the C25, Y27, and I67 sites, indicating that the I67 site is crucial for improving both substrate conversion and final product ee values ​​in the overall enzyme-catalyzed reaction.

[0198] Example 4: Combinatorial Mutation of Enone Reductase E1

[0199] 4.1 Construction of the E1 combinatorial mutant of enone reductase

[0200] To further improve the substrate conversion rate and the ee value of the final product during the catalytic reaction of the enone reductase mutant, the most effective I67 mutations (I67A and I67V) were combined with C25 mutations (C25A, C25G, and C25S) to construct combined mutants of enone reductase E1. Primers were designed based on the nucleotide sequence of wild-type enone reductase E1 (SEQ ID NO:2), and the primer sequences are shown in Table 9. Using recombinant plasmids pET28a-E1-1 (containing the C25A mutant gene), pET28a-E1-5 (containing the C25G mutant gene), and pET28a-E1-15 (containing the C25S mutant gene) as templates, combined mutants with mutations at the target sites were constructed according to the method described in Example 3.

[0201] Table 9 Primer sequences for enone reductase E1 combinatorial mutations

[0202]

[0203] 4.2 Comparison of catalytic activities of enone reductase E1 combined mutant enzymes

[0204] Following the method described in Example 2, enzyme-catalyzed reaction systems containing various combined mutants and ketone reductase E4 were constructed as shown in Table 2, and the substrates were catalyzed accordingly. Gas chromatography was used to detect the final reaction solution (i.e., the organic phase obtained after extraction with ethyl acetate from the reaction solution at the end of the reaction) of the enzyme-catalyzed reaction system, and the substrate conversion rate and the ee value of the final product were calculated. The results are shown in Table 10. When the combined mutants of ketone reductase E4 catalyzed the reaction, the substrate conversion rate exceeded 93%, and the ee value of the final product exceeded 87%. Among them, the C25G / I67A (i.e., E1-60) and C25A / 167A (i.e., E1-58) mutants showed the best catalytic effect, with ee values ​​of the final product (92.22% and 91.48%, respectively) higher than those of the single mutants.

[0205] Table 10. Enzyme sequences and catalytic effects of combined mutants of enone reductase E1.

[0206]

[0207] Example 5: Mutation of key amino acids in enone reductase E2

[0208] The enone reductase E2 of the present invention is the same enzyme as the enone reductase 2 described in the patent application document with application number 202310791779.2. The mutant enzymes of enone reductase E2 in Table 11 are also the same as the mutant enzymes of enone reductase 2 described in 202310791779.2.

[0209] Following the method described in Example 2, enzyme-catalyzed reaction systems containing each mutant enzyme from Table 11 and ketone reductase E4 were constructed as shown in Table 2, and the substrates were catalyzed accordingly. Gas chromatography was used to detect the final reaction solution (i.e., the organic phase obtained after extraction with ethyl acetate from the reaction solution at reaction termination) of the enzyme-catalyzed reaction system, and the substrate conversion rate and the ee value of the final product were calculated to evaluate the catalytic activity of each mutant enzyme on the substrate. The results are shown in Table 11. Compared with the wild-type enzyme, the mutant enzymes E2-1 (W117A mutant), E2-2 (Y83W / W117A mutant), and E2-3 (T38S / Y83W / W117A mutant) of enone reductase E2 showed improved substrate conversion and ee value of the final product during the catalytic reaction. The mutant enzymes E2-4 (T38S / Y83W / W117A / P296F) and E2-5 (L37A / T38S / Y83W / W117A / P296F) also showed a significant increase in substrate conversion during the catalytic reaction. Among them, enzyme E2-1 with only the W117A mutation showed the best catalytic effect, with an ee value of 88.11% for the final product.

[0210] Based on the sequence alignment and three-dimensional structure analysis of enone reductases, the T38 and Y83 sites of enone reductase E2 are homologous to the C25 and I67 sites of enone reductase E1, respectively. The positions 38 and 83 are referenced to the amino acid residue numbers in SEQ ID NO:3, and the positions 25 and 67 are referenced to the amino acid residue numbers in SEQ ID NO:1.

[0211] Table 11 Enzyme sequences and catalytic effects of the E2 mutant enone reductase

[0212]

[0213] Example 6: Mutation of key amino acids in enone reductase E3

[0214] The enone reductase E3 of the present invention is the same enzyme as the enone reductase 3 described in the patent application document with application number 202310791779.2. The mutants of enone reductase E3 in Table 12 are also the same as the mutants of enone reductase 3 described in 202310791779.2.

[0215] Following the method described in Example 2, enzyme-catalyzed reaction systems containing each of the mutant enzymes in Table 12 and ketone reductase E4 were constructed as shown in Table 2, and the substrates were catalyzed accordingly. Gas chromatography was used to detect the final reaction solution (i.e., the organic phase obtained after extraction with ethyl acetate from the reaction solution at the end of the reaction) of the enzyme-catalyzed reaction system, and the substrate conversion rate and the ee value of the final product were calculated to evaluate the catalytic activity of each mutant enzyme on the substrate. The results are shown in Table 12. Compared with the wild-type enzyme, the mutant enzyme of ketone reductase E3 showed significantly improved substrate conversion rate and the ee value of the final product during the catalytic reaction. Among them, enzyme E3-1 with the W103A single mutation showed the best effect, with an ee value of 88.24%.

[0216] Based on the sequence alignment and three-dimensional structure analysis of enone reductases, the T28 and Y69 sites of enone reductase E3 are homologous to the C25 and I67 sites of enone reductase E1, respectively. The amino acid residues at positions 28 and 69 are referenced from the amino acid residue numbers in SEQ ID NO:5, and positions 25 and 67 are referenced from the amino acid residue numbers in SEQ ID NO:1.

[0217] Table 12 Sequences and catalytic effects of enone reductase E3 mutants

[0218]

[0219] Example 7: Scale-up of the one-pot preparation of (R)-4-propyl-dihydrofuran-2-one

[0220] 7.1 Preparation of crude enzyme solution

[0221] Recombinant *E. coli* were cultured and induced according to the method described in Example 1 to obtain wet cells expressing enone reductase E1-55, ketone reductase E4, and glucose dehydrogenase E6, respectively. 100 g of each wet cell was taken and resuspended in 0.4 L of 100 mM PBS buffer (pH 7.5) (i.e., homogenization ratio 1 g: 4 mL), and homogenized four times at 650 bar. After centrifugation at 5000 rpm for 25 min, the precipitate was discarded, and the supernatant was collected to obtain the crude enzyme solution, which was stored at 4 °C for later use.

[0222] 7.2 One-pot scale-up reaction

[0223] Add 339g of water, 12mL of crude enone reductase E1-55 solution, 12mL of crude ketone reductase E4 solution, 12mL of crude glucose dehydrogenase E6 solution, and 0.6g of NADPNa2 to a 1L three-necked flask. Preheat the flask to an internal temperature of 20℃ with stirring, and maintain the pH at 7.5 throughout the process using 5% sodium hydroxide. Dissolve 79.8g of glucose in 125g of water and add it to the reaction solution at a uniform rate over 4 hours using a peristaltic pump; simultaneously, add 30g of 5-hydroxy-4-n-propyl-2(5H)-furanone to the reaction solution at a uniform rate over 4 hours using a peristaltic pump. While adding the materials, maintain the pH at 7.3–7.5, and continue the reaction for 2 hours at an internal temperature of 20℃ (i.e., the total reaction time is 6 hours). After the reaction was complete, 0.5 mL of the reaction solution was taken, and the pH was adjusted to <2.0 with HCl. The solution was then incubated in a water bath at 70℃ for 30 min. After extraction with ethyl acetate, the reaction solution was detected by gas chromatography. The conversion rate of the substrate and the ee value of the final product were obtained based on the detection results, as shown in Table 13.

[0224] Table 13 Results of the one-pot scale-up reaction

[0225] Reaction system numbering Enzyme composition of the reaction system substrate conversion rate ee value of the final product Reaction system B1 E1-55, E4, E6 90.77％ 92.21％

[0226] As shown in Table 13, when carrying out a one-pot scale-up reaction, a high substrate conversion rate and a high ee value of the final product can be achieved in a shorter reaction time, which is beneficial for industrial production.

[0227] sequence:

[0228] SEQ ID NO:1 (Amino acid sequence of wild-type enone reductase E1 from Thermoanaerobacter)

[0229] MSILHMPLKIKDITIKNRIMMSPMCMYSASTDGMPNDWHIVHYATRAIGGVGLIMQEATAVESRGRITDHDLGIWNDEQVKELKKIVDICKANGAVMGIQLAHAGRKCNISYEDVVGPSPIKAGDRYKLPRELSVEEIKSIVKAFGEAAKRANLAGYDVVEIHAAHGY LIHEFLSPLSNKRKDEYGNSIENRARFLIEVIDEVRKNWPENKPIFVRVSADDYMEGGINIDMMVEYINMIKDKVDLIDVSSGGLLNVDINLYPGYQVKYAETIKKRCNIKTSAVGLITTQELAEEILSNERADLVALGRELLRNPYWVLHTYTSKEDWPKQYERAFKK

[0230] SEQ ID NO:2 (nucleotide sequence of wild-type enone reductase E1 from Thermoanaerobacter)

[0231]

[0232] SEQ ID NO:3 (Amino acid sequence of wild-type enone reductase E2 from Saccharomyces cerevisiae S288C)

[0233] MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTK IFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLN QFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0234] SEQ ID NO:4 (nucleotide sequence of wild-type enone reductase E2 from Saccharomyces cerevisiae S288C)

[0235]

[0236] SEQ ID NO:5 (Amino acid sequence of wild-type enone reductase E3 from Chroococcidiopsis thermalis)

[0237] MNTNIDLFSPVRLGRYELPNRMVMAPLTRNRAGEGNVPRELNAEYYAQRVSAGLIITEQVSPQGLGYPFTPGIHSQEQVEGWRLVTKAVHDRGGKIFLQLWHVGRISHPDLQVDGALPVAPSAIAPSEGMAATYEGEKPYVTPRALETAEPGIVEQYRQGAKNALAAGFDGVEIHSANGYLL DQFLHDGSNHRTDEYGGSIENRARLLMEVTEAVVSVWGADRVGVRLSPSGTFGSVYDSDLKALFTYVVDALNQFELAYLHLVEPRVAGNETVENPTSELSSKYFRPIYKGTLISAGGYDRESGNAVLASGDADLVAYGRLFISNPDLPQRFALNAQLNPYDRSSFYGGDKRGYTDYPSLELQAAG

[0238] SEQ ID NO:6 (nucleotide sequence of wild-type enone reductase E3 from Chroococcidiopsis thermalis)

[0239]

[0240] SEQ ID NO:7 (Amino acid sequence of ketone reductase E4 from Geobacillus sp. A8)

[0241] MRLKGKAAIVTGGASGIGRATAAIRFAEEGAKVAVSDINEEGGEETVRLIREKGGEAIFVQTDVADSKQVSRLVQTAVDAFGGLHILFNNAGIGHSEVRSTDLSEEEWDRVINVNLKGVFLGIKYA VPVMKQCGGGAIVNTSSLLGIKGKKYESAYNASKAGVILLTKNAALEYGKFNIRVNAIAPGVIDTNIITPWKQDERKWPIISKANALGRIGTPEEVANAVLFLASDEASFITGATLSVDGGGLTF

[0242] SEQ ID NO:8 (nucleotide sequence of ketone reductase E4 from Geobacillus sp. A8)

[0243] ATGCGTCTGAAAGGCAAAGCGGCGATCGTTACCGGCGGCGCGAGCGGCATCGGTCGTGCGACCGCGATCCGTTTCGCTGAAGAAGGTGCGAAAGTTGCGGTTAGCGATATCAACGAAGAAGGCGGTGAAGAAACCGTTCGTCTGATCCGTGAAAAAGGCGGTGAAGCGATCTTCGTTCAGACCGATGTTGCGGATTCTAAACAGGTTTCTCGTCTGGTTCAGACCGCGGTTGATGCGTTCGGCGGTCTGCATATCCTGTTCAACAACGCGGGTATCGGCCACAGCGAAGTTCGTTCTACCGATCTGAGCGAAGAAGAATGGGACCGTGTTATCAACGTGAACCTGAAAGGCGTTTTCCTGGGCATCAAATATGCGGTTCCGGTGATGAAACAGTGCGGCGGCGGCGCTATCGTTAACACCTCTAGCCTGCTGGGTATCAAAGGCAAAAAATACGAAAGCGCGTACAACGCGTCCAAAGCGGGTGTTATCCTGCTGACCAAAAACGCGGCGCTGGAATATGGTAAATTCAACATCCGTGTTAACGCGATCGCGCCGGGCGTTATCGACACCAACATCATCACCCCGTGGAAACAGGATGAACGTAAATGGCCGATCATCAGCAAAGCGAACGCGCTGGGCCGCATCGGCACCCCGGAAGAAGTTGCGAACGCGGTTCTGTTCCTGGCGTCTGATGAAGCGAGCTTCATCACCGGCGCGACCCTGAGCGTTGATGGTGGTGGCCTGACCTTCTAA

[0244] SEQ ID NO:9 (Amino acid sequence of alcohol dehydrogenase E5 derived from Lactobacillus brevis KB290)

[0245] MEVVQMSNRLDGKVAIVTGGTLGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTR LGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0246] SEQ ID NO:10 (nucleotide sequence of alcohol dehydrogenase E5 from Lactobacillus brevis KB290)

[0247] Atggaggtcgttcaaatgtcaaaccggttagatggaaaagtagcaatcgttacaggtggtacgttgggtatcggtttagctatcgccacgaagttcgttgaagaaggggctaaggtcatgattaccggccggcacagcgatgttggtgaaaaagcagctaagagtgtcggcactcctgatcagattcaatttttccaacatgattcttccgatgaagacggctggacgaaattattcgatgcaacggaaaaagcctttggcccagtttctacattagttaataacgctgggatcgcggttaacaagagtgtcgaagaaaccacgactgctgaatggcgtaaactattagccgtcaaccttgatggtgtcttcttcggtacccgattagggattcaacggatgaagaacaaaggcttaggggcttccatcatcaacatgtcttcgatcgaaggctttgtgggtgatcctagcttaggggcttacaacgcatctaaaggggccgtacggattatgtccaagtcagctgccttagattgtgccctaaaggactacgatgttcgggtaaacactgttcaccctggctacatcaagacaccattggttgatgacttaccaggggccgaagaagcgatgtcacaacggaccaagacgccaatgggccatatcggtgaacctaacgatattgcctacatctgtgtttacttggcttctaacgaatctaaatttgcaacgggttctgaatttgtagttgatggtggttataccgctcaataa

[0248] SEQ ID NO:11 (Amino acid sequence of glucose dehydrogenase E6 derived from Bacillus subtilis 168)

[0249] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0250] SEQ ID NO:12 (nucleotide sequence of glucose dehydrogenase E6 from Bacillus subtilis 168)

[0251] ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCTATTACAGGAGCTGCTTCAGGGCTCGGAAAGGCGATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTATTATAGTAATAAACAAGATCCGAACGAGGTAAAAGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGATGTCACGAAAGAGGAAGATGTAAAAAATATCGTGCAAACGGCAATTAAGGAGTTCGGCACACTCGATATTATGATTAATAATGCCGGTCTTGAAAATCCTGTGCCATCTCACGAAATGCCGCTCAAGGATTGGGATAAAGTCATCGGCACGAACTTAACGGGTGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTCGTAGAAAACGATATCAAGGGAAATGTCATTAACATGTCCAGTGTGCACGAAGTGATTCCTTGGCCGTTATTTGTCCACTATGCGGCAAGTAAAGGCGGGATAAAGCTGATGACAGAAACATTAGCGTTGGAATACGCGCCGAAGGGCATTCGCGTCAATAATATTGGGCCAGGTGCGATCAACACGCCAATCAATGCTGAAAAATTCGCTGACCCTAAACAGAAAGCTGATGTAGAAAGCATGATTCCAATGGGATATATCGGCGAACCGGAGGAGATCGCCGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATCACGTTATTCGCGGACGGCGGTATGACACAATATCCTTCATTCCAGGCAGGCCGCGGTTAA

Claims

1. A ketene reductase, characterized in that, The amino acid sequence of the ketone reductase has at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:1, and there are at least one or more differences among amino acid residues I67, C25, Y27 and their homologous sites, wherein the positions of 67, 25 and 27 refer to the amino acid residue numbers in SEQ ID NO:

1. Preferably, the amino acid residue differences of I67 are I67A, I67C, I67D, I67E, I67F, I67G, I67H, I67K, I67L, I67M, I67N, I67P, I67Q, I67R, I67S, I67T, I67V, I67W, or I67Y; and / or, the amino acid residue differences of C25 are C25A, C25D, C25E, C25F, C25Y, C25E, C25F, C25Y, C25E, C25F, C25Y, C25E, C25Y ... 25G, C25H, C25I, C25K, C25N, C25P, C25Q, C25R, C25S, C25T, C25W or C25Y; and / or, the amino acid residues of said Y27 differ from those of Y27A, Y27C, Y27D, Y27E, Y27F, Y27H, Y27I, Y27K, Y27N, Y27P, Y27Q, Y27R, Y27S, Y27V or Y27W.

2. The enone reductase according to claim 1, characterized in that, The amino acid sequence of the ketone reductase has the following amino acid residues compared to the amino acid sequence shown in SEQ ID NO:1: I67A, I67C, I67D, I67E, I67F, I67G, I67H, I67K, I67L, I67M, I67N, I67P, I67Q, I67R, I67S, I67T, I67V, I67W, I67Y, C25A, C25D, C25E, C25F, C25G, C25H, C25I, C25K, C25N, C25 Differences between P, C25Q, C25R, C25S, C25T, C25W, C25Y, Y27A, Y27C, Y27D, Y27E, Y27F, Y27H, Y27I, Y27K, Y27N, Y27P, Y27Q, Y27R, Y27V, Y27W, C25A / I67A, C25A / I67V, C25G / I67A, C25G / I67V, C25S / I67A, or C25S / I67V; Preferably, the amino acid sequence of the enone reductase differs from the amino acid sequence shown in SEQ ID NO:1 in the following amino acid residues: I67A, I67V, I67C, I67D, I67F, I67G, I67H, I67L, I67N, I67P, I67R, I67S, I67T, I67Y, C25A, C25G, C25K, C25N, C25S, C25Y, C25A / I67A, C25A / I67V, C25G / I67A, C25G / I67V, C25S / I67A, or C25S / I67V.

3. A multi-enzyme system, characterized in that, The multi-enzyme system comprises enone reductase and ketone reductase; the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase according to claim 1 or 2; the amino acid sequence of the ketone reductase is as shown in SEQ ID NO:

7. Preferably, the multi-enzyme system further comprises dehydrogenases, wherein the dehydrogenases are formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; More preferably, the amino acid sequence of the alcohol dehydrogenase is as shown in SEQ ID NO:9; and / or, the amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO:

11.

4. An isolated nucleic acid or nucleic acid composition, characterized in that, The nucleic acid encodes the enone reductase as described in claim 1 or 2; the nucleic acid composition is composed of nucleic acids encoding enzymes in the multi-enzyme system as described in claim 3; Preferably, the nucleotide differences corresponding to the amino acid residue differences of the enone reductase are as follows: ATT67GCT, ATT67TGT, ATT67GAT, ATT67GAG, ATT67TTT, ATT67GGT, ATT67CAT, ATT67AAG, ATT67CTG, ATT67ATG, ATT67AAT, ATT67CCG, ATT67CAA, ATT67CGT, ATT67AGT, ATT67ACT, ATT67GTT, ATT67TGG, ATT67TAT, TGC25GCG, TGC25GAT, TGC25GAA, TGC25TTT, TG C25GGA, TGC25CAT, TGC25ATT, TGC25AAG, TGC25AAT, TGC25CCG, TGC25CAA, TGC25AGA, TGC25AGC, TGC25ACG, TGC25TGG, TGC25TAT, TAT27GCG, TAT27 TGT, TAT27GAT, TAT27GAG, TAT27TTT, TAT27CAT, TAT27ATT, TAT27AAG, TAT27AAT, TAT27CCT, TAT27CAG, TAT27CGT, TAT27AGT, TAT27GTG and TAT27TGG.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid or nucleic acid composition as described in claim 4; Preferably, the recombinant expression vector comprises a pET28a plasmid backbone.

6. A transformant, characterized in that, The transformant comprises: a host cell; and, an isolated nucleic acid or nucleic acid composition as described in claim 4, introduced into the host cell, or, a recombinant expression vector as described in claim 5; Preferably, the host cells include eukaryotic cells and prokaryotic cells; More preferably, the host cell is bacteria; More preferably, the bacteria is Escherichia coli, such as Escherichia coli BL21(DE3).

7. A method for preparing intermediate product 1, characterized in that, The method includes: in the presence of a cofactor, using enone reductase to catalyze the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone to obtain the intermediate product 1; The intermediate product 1 is a compound as shown in Formula 1: Preferably, the method includes one or more of the following conditions: (1) The amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or has a difference of amino acid residue Y27S compared with the amino acid sequence shown in SEQ ID NO:1, or the enone reductase is the enone reductase as described in claim 1 or 2. (2) The cofactor is NADPH and / or NADH; (3) The concentration of the 5-hydroxy-4-n-propyl-2(5H)-furanone added in the reaction is 2-80 mg / mL, preferably 5-60 mg / mL, for example 10 mg / mL or 60 mg / mL; (4) When calculating the added mass of the enone reductase based on the mass of the wet bacterial cells that produce the enone reductase, the ratio of the added mass of the enone reductase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.02-2.5):1, preferably (0.05-2.2):1, for example 0.1:1; More preferably, the cofactor is obtained through the following cofactor regeneration step: reduction of NADP in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + ; More preferably, the method includes one or more of the following conditions: (1) The NADP + and / or NAD + The mass ratio of the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.01-0.5):1, preferably (0.015-0.4):

1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1; (2) The dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formate or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol. (3) When calculating the added mass of the dehydrogenase based on the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1; (4) When the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%; when the hydrogen donor is glucose, the molar ratio of the added glucose to the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (1-5):1, preferably (1-3):1, for example 2.7:1 or 2.1:

1.

8. A method for preparing intermediate product 2, characterized in that, The method includes: in the presence of a cofactor, using a ketone reductase to catalyze the reaction of intermediate 1 to obtain intermediate 2; The intermediate product 1 is a compound as shown in Formula 1: The intermediate product 2 is a compound as shown in Formula 2: Preferably, the method includes one or more of the following conditions: (1) The amino acid sequence of the ketone reductase is shown in SEQ ID NO:7; (2) The cofactor is NADPH and / or NADH; (3) The concentration of intermediate product 1 added to the reaction system is 2-80 mg / mL, preferably 5-60 mg / mL, for example 10 mg / mL or 60 mg / mL; (4) When calculating the added mass of the ketone reductase based on the mass of the wet bacterial cells that produce the ketone reductase, the ratio of the added mass of the ketone reductase to the added mass of the intermediate product 1 is (0.05-10):1, preferably (0.1-5):1, for example 2:1 or 0.1:1; More preferably, the cofactor is obtained through the following cofactor regeneration step: reduction of NADP in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + ; More preferably, the method includes one or more of the following conditions: (1) The NADP + and / or NAD + The mass ratio of the intermediate product 1 to the added intermediate product is (0.01-0.5):1, preferably (0.015-0.4):

1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1; (2) The dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formate or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol. (3) When calculating the added mass of the dehydrogenase based on the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to the added mass of the intermediate product 1 is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1; (4) When the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%; when the hydrogen donor is glucose, the molar ratio of glucose to intermediate 1 is (1-5):1, preferably (1-3):1, for example 2.7:1 or 2.1:1; (5) The intermediate product 1 is prepared by the method as described in claim 7.

9. A method for preparing buvastan intermediate (R)-4-propyl-dihydrofuran-2-one, characterized in that, The method includes: Intermediate product 1 was obtained by catalyzing the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone with enone reductase, and (R)-4-propyl-dihydrofuran-2-one was prepared from said intermediate product 1. The intermediate product 1 is a compound as shown in Formula 1: Preferably, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in claim 1 or 2; and / or, The method for obtaining intermediate 1 by using enone reductase to catalyze the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone to obtain intermediate 1 is as described in claim 7.

10. The method as described in claim 9, characterized in that, The preparation of (R)-4-propyl-dihydrofuran-2-one using the intermediate product 1 specifically includes: using ketone reductase to catalyze the reaction of intermediate product 1 to obtain intermediate product 2; intermediate product 2 undergoes a ring-closing reaction and is finally converted into the (R)-4-propyl-dihydrofuran-2-one. The intermediate product 2 is a compound as shown in Formula 2: Preferably, the amino acid sequence of the ketone reductase is as shown in SEQ ID NO:7; and / or, The method for obtaining intermediate 2 by catalyzing the reaction of intermediate 1 with ketone reductase is as described in claim 8.

11. A method for preparing buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one, characterized in that, The method includes: Intermediate product 1 was reacted with ketone reductase to obtain intermediate product 2; intermediate product 2 was eventually converted into (R)-4-propyl-dihydrofuran-2-one through a ring-closure reaction. The intermediate product 1 is a compound as shown in Formula 1: The intermediate product 2 is a compound as shown in Formula 2: Preferably, the amino acid sequence of the ketone reductase is as shown in SEQ ID NO:7; and / or, The method for obtaining intermediate 2 by catalyzing the reaction of intermediate 1 with ketone reductase is as described in claim 8.

12. The method as described in claim 11, characterized in that, The method further includes: using enone reductase to catalyze the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone to obtain the intermediate product 1; Preferably, the amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in claim 1 or 2; and / or, The method for obtaining intermediate 1 by using enone reductase to catalyze the reaction of 5-hydroxy-4-n-propyl-2(5H)-furanone is as described in claim 7.

13. A method for preparing buvastan intermediate (R)-4-propyl-dihydrofuran-2-one, characterized in that, The method includes: in the presence of a cofactor, 5-hydroxy-4-n-propyl-2(5H)-furanone reacts under the catalysis of enone reductase and ketone reductase to generate intermediate product 2, and then undergoes a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one. The intermediate product 2 is a compound as shown in Formula 2: Preferably, the method includes one or more of the following conditions: (1) The amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or has a difference of amino acid residue Y27S compared with the amino acid sequence shown in SEQ ID NO:1, or the enone reductase is the enone reductase as described in claim 1 or 2. (2) The amino acid sequence of the ketone reductase is shown in SEQ ID NO:7; (3) The 5-hydroxy-4-n-propyl-2(5H)-furanone is added by adding it to a mixture containing the enone reductase and the ketone reductase over a period of time. (4) The ratio of the added mass of 5-hydroxy-4-n-propyl-2(5H)-furanone to the final total volume of the reaction system is 2-80 mg:1 mL, preferably 5-60 mg:1 mL, for example 10 mg:1 mL or 60 mg:1 mL. (5) When calculating the added mass of the enone reductase based on the mass of the wet bacterial cells that produce the enone reductase, the ratio of the added mass of the enone reductase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.02-2.5):1, preferably (0.05-2.2):1, for example 0.1:1; (6) When calculating the added mass of the ketone reductase based on the mass of the wet bacterial cells that produce the ketone reductase, the ratio of the added mass of the ketone reductase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-10):1, preferably (0.1-5):1, for example 2:1 or 0.1:1; (7) The cofactor is NADPH and / or NADH; More preferably, the addition is carried out at a constant rate; and / or, The NADPH and / or NADH are obtained through the following cofactor regeneration step: reduction of NADP in the presence of dehydrogenase and hydrogen donor. + and / or NAD + ; More preferably, the method includes one or more of the following conditions: (1) The NADP + and / or NAD + The mass ratio of the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.01-0.5):1, preferably (0.015-0.4):

1. For example, when adding NADP... + When the ratio of the added mass is 0.37:1 or 0.019:1; (2) The addition rate of the 5-hydroxy-4-n-propyl-2(5H)-furanone is 5-9 g / h, for example 7.5 g / h; (3) The dehydrogenase is formate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and / or glucose dehydrogenase; the hydrogen donor is formate or formate salt, D-glucose-6-phosphate, C1-C3 fatty alcohol and / or glucose, wherein the C1-C3 fatty alcohol is preferably isopropanol and / or ethanol. (4) When calculating the added mass of the dehydrogenase based on the mass of the wet bacterial cells that produce the dehydrogenase, the ratio of the added mass of the dehydrogenase to that of the 5-hydroxy-4-n-propyl-2(5H)-furanone is (0.05-6):1, preferably (0.1-1):1, for example 0.6:1 or 0.1:1; (5) When the hydrogen donor is a C1-C3 fatty alcohol, the added volume of the C1-C3 fatty alcohol accounts for 2%-25% of the total volume of the reaction, preferably 4%-20%, for example 6%; when the hydrogen donor is glucose, the molar ratio of the added glucose to the added 5-hydroxy-4-n-propyl-2(5H)-furanone is (2-6):1, preferably (2-4):1, for example 2.7:1 or 2.1:

1.

14. The method according to any one of claims 7-13, characterized in that, The method includes one or more of the following conditions: (1) The pH value of the reaction is 5-8; (2) The reaction temperature is 20-35℃; (3) The reaction time is 0.5-21 h; (4) The closed-loop reaction is carried out under acidic and heating conditions; Preferably, the method includes one or more of the following conditions: (1) The pH value is adjusted by a buffer solution or an acid-base solution, wherein the buffer solution is preferably PBS buffer, and the acid-base solution is preferably hydrochloric acid solution or sodium hydroxide solution; (2) The pH value of the acidic conditions is <2.0; (3) The heating conditions are a 60-80℃ water bath, for example, a 70℃ water bath; (4) The duration of the heating conditions is 20-60 min, for example 30 min.

15. An enzyme reaction system, characterized in that, The enzyme reaction system includes enone reductase or a multi-enzyme system, and 5-hydroxy-4-n-propyl-2(5H)-furanone; The amino acid sequence of the enone reductase is as shown in SEQ ID NO:1 or differs from the amino acid sequence shown in SEQ ID NO:1 by the amino acid residue Y27S, or the enone reductase is the enone reductase as described in claim 1 or 2; the multi-enzyme system is the multi-enzyme system as described in claim 3. Preferably, the reaction catalyzed by the enzyme reaction system is as described in any one of claims 7, 9, 12-13.

16. An enzyme reaction system, characterized in that, The enzyme reaction system contains ketone reductase and intermediate product 1; The intermediate product 1 is a compound as shown in Formula 1: The amino acid sequence of the ketone reductase is shown in SEQ ID NO:7; Preferably, the reaction catalyzed by the enzyme reaction system is as described in any one of claims 8, 10-11.

17. Use of an amino acid sequence as shown in SEQ ID NO:1 or having a difference of amino acid residue Y27S compared to the amino acid sequence shown in SEQ ID NO:1, or an enone reductase as described in claim 1 or 2, a ketone reductase with an amino acid sequence as shown in SEQ ID NO:7, a multi-enzyme system as described in claim 3, a nucleic acid or nucleic acid composition as described in claim 4, a recombinant expression vector as described in claim 5, a transformant as described in claim 6, or an enzyme reaction system as described in claim 15 or 16 in the preparation of buvasidan intermediate (R)-4-propyl-dihydrofuran-2-one or buvasidan using 5-hydroxy-4-n-propyl-2(5H)-furanone or intermediate 1; The structure of intermediate product 1 is shown in Formula 1:

Images