Ketene reductase and method for preparing brivaracetam intermediate by using ketene reductase

Abstract

The invention discloses ketene reductase and a method for preparing a brivaracetam intermediate by using the ketene reductase. The ketene reductase has at least a difference of an amino acid residue W117 or a homologous site thereof relative to an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and the position 117 refers to an amino acid residue number in SEQ ID NO: 1. Compared with a wild type, the single mutant or the combined mutant of ketene reductase from different sources has good enantioselectivity and relatively high conversion rate when the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-ketone is produced, and a good strategy is provided for industrial production of (R)-4-propyl-dihydrofuran-2-ketone.

Description

Technical Field

[0001] The present invention belongs to the field of bioenzyme catalysis, and specifically relates to an enone reductase and a method for preparing a brivaracetam intermediate using the same, as well as a compound having a structure as shown in Formula 4, a method for preparing the same and uses thereof. Background Art

[0002] Brivaracetam is a new type of anti-epileptic drug developed by UCB of Belgium. It has a high affinity for synaptic vesicle protein 2A (SV2A) and is used as an adjuvant treatment for partial seizures with or without secondary generalized seizures in adults and adolescents over 16 years of age with epilepsy. (R)-4-propyl-dihydrofuran-2-one is its important intermediate.

[0003]

[0004] (R)-4-Propyl-dihydrofuran-2-one

[0005] There are many preparation methods for (R)-4-propyl-dihydrofuran-2-one. For example, WO2016191435 uses (R)-epichlorohydrin as a raw material to obtain (R)-4-propyl-dihydrofuran-2-one through a three-step reaction, and (R)-4-propyl-dihydrofuran-2-one is further used to obtain buvacetam.

[0006]

[0007] CN106588741A uses (R)-3-methoxycarbonylhexanoic acid as a raw material, and obtains (R)-4-propyl-dihydrofuran-2-one through cyclization. (R)-4-propyl-dihydrofuran-2-one is then subjected to ring opening, substitution, and cyclization to obtain brivaracetam.

[0008]

[0009] However, all of the above methods are chemical methods, and all use chiral raw materials, which are relatively expensive. In addition, due to the specificity and high catalytic efficiency of enzyme catalysis, the enzyme-catalyzed preparation of (R)-4-propyl-dihydrofuran-2-one has also been favored.

[0010] Ketene reductase is an NAD(P)H-dependent oxidoreductase that can catalyze the asymmetric reduction of activated carbon-carbon double bonds, thereby enantioselectively synthesizing the target product. Currently, the methods for synthesizing (R)-4-propyl-dihydrofuran-2-one using ketene reductase mostly use 4-n-propyl-2(5H)-furanone as the substrate, such as:

[0011] CN111154735B provides a method for preparing (R)-4-propyl-dihydrofuran-2-one by using ketone reductase from Candida castelii to catalyze the reduction reaction of chemically synthesized 4-n-propyl-2(5H)-furanone, and the reaction is carried out at 30°C for 24 hours, and the ee value of the target product is 99.4%. However, the reaction time of this method is long.

[0012] CN113444702B discloses a mutant OYE296A of ketone 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 of greater than 99% and an ee value of the product of greater than 99%. However, the reaction time of this method is long, and 4-propyl-2(5H)-furanone is not easy to purchase, which is not conducive to industrial production.

[0013] In summary, it is necessary to find a method that is more efficient, less costly, and more suitable for industrial production to catalytically prepare (R)-4-propyl-dihydrofuran-2-one. Summary of the invention

[0014] In order to solve the defects of low efficiency, high cost or unsuitability for industrial production in the preparation of (R)-4-propyl-dihydrofuran-2-one by enone reductase in the prior art, the present invention provides an enone reductase and a method for preparing a brivaracetam intermediate using the same. Compared with the wild type, the single mutants or combined mutants of enone reductase from different sources in the present invention all show good enantioselectivity and high conversion rate when producing the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, providing a good strategy for the industrial production of (R)-4-propyl-dihydrofuran-2-one.

[0015] To solve the above problems, the first aspect of the present invention provides an enone reductase, wherein the enone reductase has at least a difference in amino acid residue W117 or its homologous site relative to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or at least a difference in amino acid residue W117 or its homologous site relative to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 having at least 95%, 96%, 97%, 98% or 99% identity; wherein position 117 is numbered with reference to the amino acid residue in SEQ ID NO:1. Herein, identity refers to the degree of sequence consistency obtained when amino acid sequences are aligned. The method for calculating identity is: first determine the amino acid sequence as a comparison benchmark, and then compare the sequence consistency of another amino acid sequence with the amino acid sequence as a comparison benchmark, and identity = the number of amino acids that are exactly the same after the two amino acid sequences are aligned ÷ the total number of amino acids in the amino acid sequence as a comparison benchmark × 100%. Homologous sites refer to the corresponding sites when amino acid sequences are aligned.

[0016] In some embodiments, the enone reductase is selected from the group consisting of:

[0017] (1) The ketone reductase has a difference selected from the group consisting of amino acid residues W117A, W117S, W117P, W117G, W117V, W117C, W117R, W117E, W117K, W117L, W117I or W117T relative to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; preferably, it has a difference in amino acid residue W117A;

[0018] More preferably, the ketene reductase has one or more of the following differences in amino acid residues: L37A, T38S / L, Y83W and P296F relative to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, wherein positions 37, 38, 83 and 296 are numbered with reference to the amino acid residues in SEQ ID NO: 1 or SEQ ID NO: 3; or,

[0019] (2) The ketene reductase has a difference selected from the amino acid residues W103A, W103S, W103P, W103G, W103V, W103C, W103R, W103E, W103K, W103L, W103I or W103T relative to the amino acid sequence shown in SEQ ID NO:5; preferably, it has a difference in amino acid residue W103A; W103 of the amino acid sequence shown in SEQ ID NO:5 and W117 of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 are homologous sites. Herein, the " / " used for the difference in amino acid residues at the same site represents that there are multiple choices of amino acid residues at that site.

[0020] More preferably, the ketene reductase further has one or more of the differences in amino acid residues L27A, T28S, Y69W and E275F relative to the amino acid sequence shown in SEQ ID NO: 5. Herein, L27, T28, Y69 and E275 of the amino acid sequence shown in SEQ ID NO: 5 are homologous sites to L37, T38, Y83 and P296 of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, respectively.

[0021] In some preferred embodiments, the ketene reductase further has one or more of the following differences in amino acid residues P35C / E / A / L, M40A / F / L, I76L / V, L119I, R244A, S246T and I352L relative to the amino acid sequence shown in SEQ ID NO:1.

[0022] In some specific embodiments, the ketone reductase has a difference in amino acid residues selected from the following group relative to the amino acid sequence shown in SEQ ID NO: 1:

[0023] (1)W117N, W117S, W117Q, W117P, W117G, W117D, W117V, W117C, W117E, W117Y, W117 K, W117L, W117I, W117T, W117M, W117H, W117F, W117R, P35C / L37A / T38S / Y83W / W117 V / P296F, P35C / L37A / T38S / Y83W / W117S / P296F, P35C / L37A / T38S / Y83W / W117P / P2 96F, P35C / L37A / T38S / Y83W / W117T / P296F, P35C / L37A / T38S / Y83W / W117G / P296F;

[0024] (2)W117A, Y83W / W117A, W117A / P296F, P35C / W117A, P35L / W117A, T38S / W117A, T38L / W117A, T38S / Y83W / W117A, Y8 3W / W117A / P296F, T38S / Y83W / W117A / P296F, P35A / T38S / Y83W / W117A / P296F, L37A / T38S / Y83W / W117A / P296F, T38 S / Y83W / W117A / R244A / P296F, L37A / T38S / Y83W / W117A / P296F / I352L, P35E / L37A / T38S / Y83W / W117A / P296F, P35C / L37A / T38S / Y83W / W117A / P296F, P35C / L37A / T38S / M40A / Y83W / W117A / P296F, P35C / L37A / T38S / Y83W / W117A / L119 I / P296F, P35C / L37A / T38S / Y83W / W117A / S246T / P296F, P35C / L37A / T38S / M40A / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / M40L / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / M40F / Y83W / W117A / L119I / P296F, P35C / L37A / T38 S / I76L / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / I76V / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / Y83W / W117A / L119I / S246T / P296F, P35C / L37A / T38S / M40A / W117A / P296F or P35C / L37A / T38S / M40A / W117A / L119I / P296F; or,

[0025] The ketene reductase has the following differences in amino acid residues relative to the amino acid sequence shown in SEQ ID NO: 3:

[0026] W117A, Y83W / W117A, T38S / Y83W / W117A, T38S / Y83W / W117A / P296F or L37A / T38S / Y83W / W117A / P296F; or,

[0027] The ketene reductase has the following differences in amino acid residues relative to the amino acid sequence shown in SEQ ID NO:5:

[0028] W103A, Y69W / W103A, T28S / Y69W / W103A, T28S / Y69W / W103A, T28S / Y69W / W103A / E275F or L27A / T28S / Y69W / W103A / E275F. Herein, the " / " used between the amino acid residue differences at different sites of each enzyme represents that the amino acid residue differences at the different sites are simultaneously present on the same enzyme.

[0029] The second aspect of the present invention provides an isolated nucleic acid encoding the enone reductase according to the first aspect of the present invention.

[0030] The third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid described in the second aspect of the present invention.

[0031] In some embodiments, the recombinant expression vector comprises a pET28a plasmid backbone.

[0032] The fourth aspect of the present invention provides a genetically engineered bacterium, which comprises the isolated nucleic acid described in the second aspect of the present invention or the recombinant expression vector described in the third aspect of the present invention.

[0033] The fifth aspect of the present invention provides a method for producing ketene reductase, wherein the genetically engineered bacteria described in the fourth aspect of the present invention are inoculated into a culture medium suitable for their growth to obtain the ketene reductase.

[0034] The sixth aspect of the present invention provides a method for producing an intermediate product 1, the method comprising: mixing ketone reductase or a genetically engineered bacterium producing the ketone reductase with a substrate, and obtaining the intermediate product 1 after reaction.

[0035] The substrate is a compound as shown in Formula 1:

[0036]

[0037] Wherein, R is benzyl or C1-C4 alkyl;

[0038] The intermediate product 1 is a compound as shown in Formula 2:

[0039]

[0040] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0041] In some preferred embodiments, the ketene reductase is the ketene reductase described in the first aspect of the present invention; and / or, the genetically engineered bacteria is the genetically engineered bacteria described in the fourth aspect of the present invention.

[0042] In some more preferred embodiments, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing.

[0043] In some more preferred embodiments, the buffer is PBS buffer; and / or,

[0044] The coenzyme is nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) any one or both; and / or,

[0045] The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol.

[0046] In some more preferred embodiments, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290.

[0047] In some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:15; and / or the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:17.

[0048] The seventh aspect of the present invention provides a method for producing an intermediate product 2. The method comprises: mixing ketoreductase with an intermediate product 1, and obtaining the intermediate product 2 after reaction.

[0049] The intermediate product 1 is a compound as shown in Formula 2:

[0050]

[0051] Wherein, R is benzyl or C1-C4 alkyl;

[0052] The intermediate product 2 is a compound as shown in Formula 3:

[0053]

[0054] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0055] In some preferred embodiments, the ketoreductase is selected from the group consisting of ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina, and Geobacillus sp. A8.

[0056] In some more preferred embodiments, the amino acid sequence of the ketoreductase is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13.

[0057] In some more preferred embodiments, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing.

[0058] In some more preferred embodiments, the buffer is PBS buffer; and / or,

[0059] The coenzyme is nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) any one or both; and / or,

[0060] The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol.

[0061] In some more preferred embodiments, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290.

[0062] In some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:15; and / or the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:17.

[0063] In some more preferred embodiments, the intermediate product 1 is the intermediate product 1 obtained by the method described in the sixth aspect of the present invention.

[0064] In an eighth aspect, the present invention provides a method for producing a brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, the method comprising mixing ketone reductase or a genetically engineered bacterium producing the ketone reductase with a substrate to obtain an intermediate product 1 after reaction; and using the intermediate product 1 to prepare (R)-4-propyl-dihydrofuran-2-one;

[0065] The substrate is a compound as shown in Formula 1:

[0066]

[0067] Wherein, R is benzyl or C1-C4 alkyl;

[0068] The intermediate product 1 is a compound as shown in Formula 2:

[0069]

[0070] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0071] In some preferred embodiments, the ketene reductase is the ketene reductase as described in the first aspect of the present invention; and / or, the genetically engineered bacteria is the genetically engineered bacteria as described in the fourth aspect of the present invention.

[0072] In some preferred embodiments, the method for preparing (R)-4-propyl-dihydrofuran-2-one using the intermediate product 1 comprises: mixing ketoreductase with the intermediate product 1, and obtaining the (R)-4-propyl-dihydrofuran-2-one after reaction.

[0073] In some more preferred embodiments, the method for preparing (R)-4-propyl-dihydrofuran-2-one using the intermediate product 1 comprises: mixing the ketoreductase with the intermediate product 1 to obtain the intermediate product 2 after reaction; and the intermediate product 2 undergoes a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one.

[0074] In some more preferred embodiments, the ketoreductase is selected from the group consisting of ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina, and Geobacillus sp. A8.

[0075] In some more preferred embodiments, more preferably, the amino acid sequence of the ketoreductase is selected from SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13.

[0076] In some more preferred embodiments, the enone reductase or the genetically engineered bacteria is mixed with the substrate in a buffer; and / or, coenzymes and coenzyme circulation systems are added before, during or after the enone reductase or the genetically engineered bacteria is mixed with the substrate; and / or, coenzymes and coenzyme circulation systems are added before, during or after the ketoreductase is mixed with the intermediate 1.

[0077] In some more preferred embodiments, the buffer is PBS buffer; and / or,

[0078] The coenzyme is nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) or both; and / or,

[0079] The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol.

[0080] In some more preferred embodiments, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290.

[0081] In some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:15; and / or the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:17.

[0082] The ninth aspect of the present invention provides a method for producing the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, the method comprising: mixing ketoreductase with the intermediate 1, and reacting to obtain the (R)-4-propyl-dihydrofuran-2-one;

[0083] The intermediate product 1 is a compound as shown in Formula 2:

[0084]

[0085] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0086] In some preferred embodiments, the method comprises: mixing the ketoreductase with the intermediate 1 to obtain the intermediate 2 after reaction; and subjecting the intermediate 2 to a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one.

[0087] In some preferred embodiments, the ketoreductase is selected from the group consisting of ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina, and Geobacillus sp. A8.

[0088] In some more preferred embodiments, the amino acid sequence of the ketoreductase is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13.

[0089] In some more preferred embodiments, the method further comprises mixing ketone reductase or a genetically engineered bacterium producing the ketone reductase with a substrate to obtain the intermediate product 1 after the reaction;

[0090] The substrate is a compound as shown in Formula 1:

[0091]

[0092] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0093] In some more preferred embodiments, the enone reductase is the enone reductase described in the first aspect of the present invention; and / or, the genetically engineered bacteria is the genetically engineered bacteria described in the fourth aspect of the present invention.

[0094] In some more preferred embodiments, the enone reductase or the genetically engineered bacteria is mixed with the substrate in a buffer; and / or, coenzymes and coenzyme circulation systems are added before, during or after the enone reductase or the genetically engineered bacteria is mixed with the substrate; and / or, coenzymes and coenzyme circulation systems are added before, during or after the ketoreductase is mixed with the intermediate 1.

[0095] In some more preferred embodiments, the buffer is PBS buffer; and / or,

[0096] The coenzyme is selected from nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) any one or both; and / or,

[0097] The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol.

[0098] In some more preferred embodiments, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290.

[0099] In some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:15; and / or the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:17.

[0100] The tenth aspect of the present invention provides a method for producing a brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, comprising mixing any one or two of an enone reductase or a genetically engineered bacterium producing the enone reductase, a ketone reductase and a substrate, and reacting to obtain the (R)-4-propyl-dihydrofuran-2-one;

[0101] The substrate is a compound as shown in Formula 1:

[0102]

[0103] Wherein, R is a benzyl group or a C1-C4 alkyl group.

[0104] In some preferred embodiments, the method comprises: mixing any one or both of the enone reductase or the genetically engineered bacteria producing the enone reductase, the ketone reductase and the substrate, and reacting to obtain an intermediate 2; and subjecting the intermediate 2 to a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one.

[0105] In some preferred embodiments, the ketene reductase is the ketene reductase described in the first aspect of the present invention; and / or, the genetically engineered bacteria is the genetically engineered bacteria described in the fourth aspect of the present invention.

[0106] In some preferred embodiments, the ketoreductase is selected from the group consisting of ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina, and Geobacillus sp. A8.

[0107] In some more preferred embodiments, the amino acid sequence of the ketoreductase is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13.

[0108] In some more preferred embodiments, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing.

[0109] In some more preferred embodiments, the buffer is PBS buffer; and / or,

[0110] The coenzyme is selected from nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) any one or both; and / or,

[0111] The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol.

[0112] In some more preferred embodiments, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290.

[0113] In some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:15; and / or the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:17.

[0114] The eleventh aspect of the present invention provides a compound having a structure as shown in Formula 4:

[0115]

[0116] Wherein, R is ethyl or benzyl.

[0117] The twelfth aspect of the present invention provides a method for preparing the compound described in the eleventh aspect of the present invention, the method comprising the following steps: mixing any one of benzyl bromide or ethyl bromide with 5-hydroxy-4-n-propyl-2(5H)-furanone, and obtaining the compound after reaction.

[0118] In some preferred embodiments, the reaction is carried out in a mixed reaction system of dimethylformamide (DMF) and potassium carbonate.

[0119] In some more preferred embodiments, the reaction conditions satisfy one or more of the following conditions:

[0120] The temperature is 25 to 35°C, for example 30°C;

[0121] And / or, the time is 1.5 to 6 hours, for example 2 hours.

[0122] The thirteenth aspect of the present invention provides use of the compound described in the eleventh aspect of the present invention in the production of (R)-4-propyl-dihydrofuran-2-one.

[0123] The fourteenth aspect of the present invention provides the use of the genetically engineered bacteria described in the fourth aspect of the present invention in the production of ketone reductase or (R)-4-propyl-dihydrofuran-2-one, or the use of the ketone reductase described in the first aspect of the present invention in the production of (R)-4-propyl-dihydrofuran-2-one.

[0124] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0125] The reagents and raw materials used in the present invention are commercially available.

[0126] The positive and progressive effects of the present invention are:

[0127] Compared with the wild type, single mutants or combined mutants of ketone reductase from different sources showed good enantioselectivity and high conversion rate in the production of brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, providing a good strategy for the industrial production of (R)-4-propyl-dihydrofuran-2-one. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1 Schematic diagram of the synthesis route of the substrate. R is methyl, ethyl or benzyl.

[0129] Figure 2 This is the gas chromatography detection spectrum of methyl ester substrate a.

[0130] Figure 3 For methyl ester substrate a 1 H NMR detection spectrum.

[0131] Figure 4 This is the gas chromatography detection spectrum of ethyl ester substrate b.

[0132] Figure 5 For ethyl ester substrate b 1 H NMR detection spectrum.

[0133] Figure 6 This is the gas chromatography detection spectrum of benzyl ester substrate c.

[0134] Figure 7 For benzyl ester substrate c 1 H NMR detection spectrum.

[0135] Figure 8The schematic diagram of the two-step process route for preparing (R)-4-propyl-dihydrofuran-2-one is shown in the figure. Among them, OYE (oldyellow enzyme): old yellow enzyme, also called enoate reductase in the present invention; KRED: ketoreductase; GDH: glucose dehydrogenase; Glc: glucose; Glc Ac: gluconic acid. R is methyl, ethyl or benzyl.

[0136] Fig. 9 The schematic diagram of the one-pot process for preparing (R)-4-propyl-dihydrofuran-2-one is shown in Figure 1. OYE: enone reductase; KRED: ketoreductase; IPADH: isopropanol dehydrogenase; IPA: isopropanol; Acetone: acetone. R is methyl, ethyl or benzyl. DETAILED DESCRIPTION

[0137] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0138] Unless otherwise specified, the various materials and reagents used in the examples of the present invention are commonly used materials and reagents in the art and can be obtained through conventional commercial channels.

[0139] Preparation of TB culture medium: peptone 10g / L, yeast powder 18g / L, glycerol 4mL / mL, KH2PO4 (anhydrous) 2.31g / L, K2HPO4 (trihydrate) 16.43g / L, dissolve with reverse osmosis water (RO water) and make up to volume, sterilize at 121℃ for 20min, and set aside.

[0140] Preparation of LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, dissolve with RO water and make up to volume, sterilize at 121°C for 20 min, and set aside.

[0141] The reference substance of the final product (R)-4-propyl-dihydrofuran-2-one was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., item number BD626939-1g; the reference substance of the racemate of the final product was synthesized with reference to CN109134406A. The synthesis method of the substrate is shown in Example 1, and the compound 1 (5-hydroxy-4-n-propyl-2(5H)-furanone) in Example 1 was synthesized with reference to WO2017076738A1.

[0142] The gas chromatography analysis method in the embodiment is as follows:

[0143] 1. Chromatographic conditions for the reaction of methyl ester substrate a and ethyl ester substrate b: Agilent 7820A gas chromatograph (with FID detector); chromatographic column LIPODEX E (25m×0.25mm×1μm); flow rate: 0.7mL / min; split ratio: 30:1; injection volume: 0.5μL; injection port temperature: 220℃; detector temperature: 240℃; column oven temperature: initial temperature is 130℃, maintained for 20min; diluent: methanol. A reference substance is required for gas chromatography detection, and its preparation method is: weigh 20mg of the reference substance into a 10mL volumetric flask, dissolve it with diluent and dilute to the scale.

[0144] Retention time:

[0145] (1) When the substrate is methyl ester, i.e. R is methyl: substrate a 3.616 min; intermediate product 1a racemate 4.214 min and 4.337 min;

[0146] (2) When the substrate is ethyl ester, i.e. R is ethyl: substrate b 4.936 min; intermediate product 1b racemate 4.639 min and 4.833 min;

[0147] (3) The final product racemates had time 14.511min and 15.032min, and the final product (R) had time 14.350min.

[0148] 2. Chromatographic conditions for the reaction of benzyl ester substrate c: Agilent 7820A gas chromatograph (with FID detector); chromatographic column LIPODEX E (25m×0.25mm×1μm); flow rate: 0.7mL / min; split ratio: 30:1; injection volume: 0.5μL; injection port temperature: 220℃; detector temperature: 240℃; column oven temperature: initial temperature is 130℃, maintained for 16min, increased to 180℃ at a rate of 20℃ / min, maintained for 10min; diluent: methanol. A reference substance is required for gas chromatography detection, and its preparation method is: weigh 20mg of the reference substance into a 10mL volumetric flask, dissolve it with diluent and dilute to the scale.

[0149] When the product of the first step of the two-step method is tested, the ee value of the intermediate product 1c is determined based on the ee value of the final product. Take the first step reaction solution of enone reductase and benzyl ester substrate c, add 192mM glucose and NAD +5mM, ketoreductase 7 20mg / mL, glucose dehydrogenase 6mg / mL (the sources and sequences of ketoreductase and glucose dehydrogenase are shown in Table 1), adjust pH to 7.0, react at 30°C, 220rpm for 2h, adjust pH to <2.0 with hydrochloric acid (HCl), water bath at 70°C for 1h, take 1mL of reaction solution and add 1.5mL methyl tert-butyl ether (MTBE) for extraction, and take the supernatant for gas chromatography detection of chirality.

[0150] Retention time: 14.511 min and 15.032 min for the final product racemate, 14.350 min for the final product (R).

[0151] Example 1: Chemical synthesis of substrate

[0152] The synthetic route of the substrate is Figure 1 As shown. Add 300 mesh potassium carbonate (1.1eq) and anhydrous dimethylformamide (DMF, 4V) to a three-necked jacketed bottle, and stir to dissolve at 15°C. Add compound 1 (1.0eq) and compound 2 (0.95eq), and rinse the wall with anhydrous DMF (1V), heat to 30°C for 2h, and detect whether compound 2 is residual by thin layer chromatography (TLC). If so, add compound 1 (0.05eq) and continue to react for 4h until TLC detects that compound 2 is completely reacted, and then cool to 15°C. Filter by suction, wash the filter cake with DMF (0.5V), add water (0.7V) to the filtrate and stir evenly, and extract three times with n-heptane (1.5V).

[0153] The organic phases were combined, washed with a solvent (DMF:H2O=2:1, 0.6V) to remove part of the pigment, and the separated lower layer was extracted three times with n-heptane (0.6V). All the n-heptane phases before and after were combined, washed three times with water (1V), the organic phase was dried with anhydrous sodium sulfate, and concentrated under reduced pressure with a water pump to obtain an oily substance with a yield of more than 85%. The gas phase detection spectrum and NMR detection spectrum of the methyl ester substrate a, ethyl ester substrate b, and benzyl ester substrate c are shown in Figures 2 to 7 shown.

[0154] eq means equivalent, and the number in front is the ratio of the amount of substance; V means unit volume, and the number in front is the ratio of volume.

[0155] Example 2: Acquisition of enzyme genes

[0156] 2.1 Synthesis of enzyme genes

[0157] Three enone reductases, four ketone reductases, one glucose dehydrogenase and one isopropanol dehydrogenase were screened from the NCBI database for constructing the reaction system. The sources and sequences of the enzymes are shown in Table 1. Among them, enone reductase and ketone reductase can catalyze the conversion of substrates into the final product (R)-4-propyl-dihydrofuran-2-one, and glucose dehydrogenase and isopropanol dehydrogenase can achieve the regeneration of NADH and / or NADPH in the presence of glucose and / or isopropanol, providing reducing hydrogen for the biocatalytic reduction reaction. The reaction route for preparing the final product (R)-4-propyl-dihydrofuran-2-one using the above method is as follows: Figure 8 , Fig. 9 shown.

[0158] Table 1 Enzyme sources and sequences

[0159]

[0160] The above genes were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., codon optimized and cloned into the expression vector pET28a to obtain recombinant plasmids.

[0161] 2.2 Transformation of enzyme genes

[0162] The synthesized recombinant plasmids were transformed into competent E. coli BL21 (DE3) cells, and after recovery, they were spread on LB plates containing 50 μg / mL kanamycin and cultured at 37° C. overnight.

[0163] 2.3 Enzyme gene expression

[0164] The colonies on the plate were inoculated into TB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 4 h. The colonies were inoculated into LB liquid medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum and cultured at 37°C, 250 rpm until the bacterial concentration OD 600 =0.6-0.8, add IPTG with a final concentration of 0.1 mM, induce overnight at 25°C and 250 rpm, centrifuge at 4000 rpm for 20 min, and collect the wet cells.

[0165] After obtaining the wet cells, the wet cells were resuspended in 50 mM pH 7.0 PBS buffer at a ratio of 1:10 (g / mL), homogenized with a homogenizer, and stored at -20°C for later use. The crude enzyme solutions used in the following examples were all prepared using this method.

[0166] Example 3: Comparison of catalytic activity of wild-type ketone reductases from different sources

[0167] The two-step process for preparing (R)-4-propyl-dihydrofuran-2-one is shown in Figure 8, enone reductase catalyzes the conversion of substrate into intermediate 1 as the first step.

[0168] Enone reductase catalytic reaction system: 20 mg / mL of enone reductase, 10 mg / mL of substrate, 20% (v / v) of isopropanol, 1.25 mL of 0.2M PBS buffer (pH 7.0), 5 mM NADP+, 192 mM glucose, 6 mg / mL of glucose dehydrogenase, add water to prepare 5 mL of reaction solution. The above reaction solution is placed in a 50 mL centrifuge tube, adjusted to pH 6.5 (pH 7.5 when using benzyl ester substrate c), 30°C, 220 rpm for 1 hour. Take 1 mL of the reaction solution and add it to 1 mL of ethyl acetate. After mixing, centrifuge at 12000 rpm for 5 minutes, and take the supernatant for gas chromatography detection of chirality. The substrate conversion rate and enantioselectivity of the wild-type enone reductase are shown in Table 2. The ee value is negative, indicating that the main configuration of the product is S, and the ee value is positive, indicating that the main configuration of the product is R (this representation method is used in the following examples). The conversion rates of the three wild-type enone reductases with methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c were all high (100%), but the intermediate 1 (structural formula: Figure 8 The S configuration is the main one, and further modification is needed to obtain more R-configured target products.

[0169] Table 2 Comparison of the activities of wild-type ketone reductase from three different sources

[0170]

[0171] Example 4: Saturation mutation screening of key amino acids in the active pocket of enone reductase 1

[0172] 4.1 Construction of key amino acid saturation mutants in the active pocket of enone reductase 1

[0173] Using enone reductase 1 (amino acid sequence SEQ ID NO: 1) as the starting enzyme, protein three-dimensional structure analysis software and molecular docking software were used for simulation analysis. Taking into account the docking results of enone reductase 1 and substrate, the characteristics of the enzyme's substrate binding pocket, the structural characteristics of the enzyme's stereoselective recognition of substrates, and the catalytic mechanism of the enzyme, it was finally speculated that P35, L37, T38, M40, I76, Y83, W117, L119, R244, S246, P296 and I352 sites may be sites that affect its enzyme activity. Therefore, using enone reductase 1 as the starting enzyme, single-point saturation mutations were screened for these sites.

[0174] According to the gene sequence of enone reductase 1 (SEQ ID NO: 2), the corresponding primers were designed to screen for saturation mutations at the W117 and P296 sites. The primer sequences are shown in Table 3. The pET28a plasmid containing the enone reductase 1 gene was used as a template, and PCR amplification was performed using the primers in Table 3. The PCR product was digested and purified by DpnI, and then recombined using seamless cloning technology. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and after recovery, it was spread on an LB plate containing 50 μg / mL kanamycin and cultured at 37°C overnight. After colony PCR verification and sequencing verification, a mutant with a mutation at the target site was obtained.

[0175] Table 3 Primers for saturation mutation of ketone reductase 1W117 and P296

[0176]

[0177] 4.2 Comparison of catalytic activities of saturated mutants

[0178] According to the method described in Example 3, the catalytic activity of each mutant at the W117 and P296 sites of ketone reductase 1 on methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c was evaluated under the same reaction conditions, and the chirality of the product was detected by gas chromatography. The results of the saturation mutation screening at the W117 and P296 sites are shown in Table 4 and Table 5, respectively, and the results show that the saturation mutation at the W117 position has a more obvious effect on the enantioselectivity of ketone reductase 1. Among them, the mutants of W117A, W117S, W117P, W117G, W117V, W117C, W117R, W117E, W117K, W117L, W117I and W117T have better effects. When the tryptophan at position 117 of ketone reductase 1 mutated to alanine (i.e., enzyme 1-1), the ee value of the reaction with methyl ester substrate a was -5.66%, which was significantly improved compared to the wild type. The enantioselectivity of the reaction with ethyl ester substrate b and benzyl ester substrate c was reversed, with ee values ​​of 71.78% and 72.63%, respectively, and the product changed from mainly S configuration to mainly R configuration. The conversion rates of methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c were all 100%, and the comprehensive effect of conversion rate and enantioselectivity was the best. However, the R configuration enantioselectivity of the P296 site saturation mutant was not significantly improved.

[0179] The same method was used to perform saturation mutation screening on the remaining 10 key sites of the active pocket, and no site was found that had a more significant effect on the enantioselectivity of enoketone reductase 1 than W117.

[0180] Table 4 Comparison of the activities of the W117 site saturation mutants of enone reductase 1

[0181]

[0182] Table 5 Comparison of the activities of P296 saturation mutants of enone reductase 1

[0183]

[0184] Example 5: Alanine mutation of key amino acids in ketone reductases from different sources

[0185] 5.1 Construction of alanine mutants of ketone reductase from different sources

[0186] Since the amino acid residue at position 117 is a key site affecting the enantioselectivity of ketone reductase 1, and the W117A mutant has the best catalytic activity, the homologous sites of ketone reductase 2 and ketone reductase 3 (i.e., the homologous site of ketone reductase 2 is W117, and the homologous site of ketone reductase 3 is W103) were also mutated to alanine. According to the gene sequences of ketone reductase 2 and 3 (SEQ ID NO: 4 and SEQ ID NO: 6, respectively), the corresponding primers were designed, and the primer sequences are shown in Table 6. The mutants with mutations in the target sites were constructed according to the method described in Example 4.

[0187] Table 6 Alanine mutation primers of ketone reductase from different sources

[0188]

[0189] 5.2 Comparison of catalytic activities of alanine mutants

[0190] According to the method described in Example 3, the catalytic activity of each mutant on methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c was evaluated using the same reaction conditions, and the chirality of the product was detected by gas chromatography. The results are shown in Table 7. After the W117 of enone reductase 2 and the W103 of enone reductase 3 were mutated and reacted, the configuration of the intermediate 1 was significantly changed, indicating that these two sites are indeed the key sites that affect the enantioselectivity of enone reductase 2 and enone reductase 3.

[0191] Table 7 Comparison of the activities of wild-type and alanine mutant ketone reductase from different sources

[0192]

[0193] Example 6: Combination mutations of ketone reductase 1

[0194] 6.1 Construction of combined mutants of enone reductase 1

[0195] In order to further improve the enantioselectivity of ketene reductase 1 and increase the proportion of intermediate 1 with R configuration, the 117 site and other sites were combined for mutation. According to the gene sequence of ketene reductase 1 (SEQ ID NO: 2), the corresponding primers were designed, and the primer sequences are shown in Table 8. The mutants with mutations in the target sites were constructed according to the method described in Example 4.

[0196] Table 8 Mutation primers for other sites of enone reductase 1

[0197]

[0198]

[0199] 6.2 Comparison of catalytic activities of combined mutants of enone reductase 1

[0200] According to the method described in Example 3, the catalytic activity of each mutant on methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c was evaluated under the same reaction conditions, and the chirality of the product was detected by gas chromatography. In the experiment, the inventor unexpectedly found that although the single mutation P296 of ketone reductase 1 did not show a good configuration when reacting with methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c compared with its wild type, some combined mutations of the single mutation P296F showed a good effect, that is, when the P296F mutation was present, the ee value was higher, as shown in Table 9, wherein the mutants 1-40 compared with 1-1, 1-39 compared with 1-46, and 1-45 compared with 1-47 all showed that the combined mutation of P296F could improve the ee value. Therefore, the mutation of P296F was also introduced into other mutation combinations. The final results are shown in Table 9. It can be seen that the R-configuration ee value of the combined mutants of ketone reductase 1 is further improved compared with the wild type. The ee values ​​of mutant enzyme 1-54 catalyzing the reactions of methyl ester substrate a and ethyl ester substrate b are both greater than 80%, and the ee value of catalyzing the reaction of benzyl ester substrate c is greater than 90%, and the substrate conversion rate is greater than 90%, and the effect of the reaction with the three substrates is good; mutant 1-59 catalyzes the reaction of ethyl ester substrate b with the highest ee value, reaching 84.17%; mutants 1-63 and 1-64 catalyze the best effect of benzyl ester substrate c, with conversion rates of 100% and ee values ​​of 94.45% and 92.31% respectively.

[0201] Table 9 Comparison of the activities of ketone reductase 1 combination mutants

[0202]

[0203]

[0204]

[0205] Example 7: Combination mutations of ketone reductase 2

[0206] 7.1 Construction of combined mutants of enone reductase 2

[0207] According to the partial mutation combination with better effect on enone reductase 1, the same mutation was performed on enone reductase 2. According to the gene sequence of enone reductase 2 (SEQ ID NO: 4), the corresponding primers were designed, and the primer sequences are shown in Table 10. According to the method described in Example 4, a mutant with mutation at the target site was constructed.

[0208] Table 10 Mutation primers for other sites of enone reductase 2

[0209]

[0210] 7.2 Comparison of catalytic activities of combined mutants of enone reductase 2

[0211] According to the method described in Example 3, the catalytic activity of each mutant on methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c was evaluated under the same reaction conditions, and the chirality of the product was detected by gas chromatography. The results are shown in Table 11. The R configuration ee value of the ketone reductase 2 combination mutant is further improved compared with the wild type, and except for mutant 2-5, the substrate conversion rate of all other mutants is very high (100%) when catalyzing the reaction. Mutant 2-4 has the best effect, and the ee value of catalyzing the reaction of methyl ester substrate a can reach 47.49%, the ee value of catalyzing the reaction of ethyl ester substrate b can reach 64.46%, and the ee value of catalyzing the reaction of benzyl ester substrate c can reach 75.39%.

[0212] Table 11 Comparison of the activities of ketone reductase 2 combination mutants

[0213]

[0214] Example 8: Combination mutations of ketone reductase 3

[0215] 8.1 Construction of ketone reductase 3 combined mutants

[0216] According to the partial mutation combination with better effect on enone reductase 1, the same mutation was performed on the homologous site of enone reductase 3. According to the gene sequence of enone reductase 3 (SEQ ID NO: 6), the corresponding primers were designed, and the primer sequences are shown in Table 12. The mutants with mutations in the target sites were constructed according to the method described in Example 4.

[0217] Table 12 Mutation primers for other sites of enone reductase 3

[0218]

[0219] 8.2 Comparison of catalytic activities of ketone reductase 3 combined mutants

[0220] According to the method described in Example 3, the catalytic activity of each mutant on methyl ester substrate a, ethyl ester substrate b and benzyl ester substrate c was evaluated using the same reaction conditions, and the chirality of the product was detected by gas chromatography. The results are shown in Table 13. The ee value of the R configuration of the ketone reductase 3 combination mutant was further improved compared with the wild type, and the ee value of mutants 3-4 and 3-5 when reacting with benzyl ester substrate c can reach more than 90%.

[0221] Table 13 Comparison of the activities of ketone reductase 3 combined mutants

[0222]

[0223] Example 9: Preparation of the final product (R)-4-propyl-dihydrofuran-2-one using intermediate product 1c

[0224] like Figure 8 As shown, intermediate 1 can be reduced to intermediate 2 under the catalysis of ketoreductase, and further undergoes cyclization reaction in the presence of hydrochloric acid (HCl) to generate the final product (R)-4-propyl-dihydrofuran-2-one. Since the R configuration ee value of the product when the benzyl ester substrate reacts with enone reductase is the highest among the three substrates, the benzyl ester substrate c (i.e. Figure 8 The intermediate product 1c (i.e. Figure 8 The intermediate product 1 (the R group of which is benzyl, the same below) continues to react with ketoreductase.

[0225] Ketoreductase catalytic reaction system: Take the first step reaction solution of enone reductase 1-63 and benzyl ester substrate c in Example 6, add 192mM glucose, 5mM NAD+, 20mg / mL ketoreductase, 6mg / mL glucose dehydrogenase, adjust pH to 7.0, react at 30°C, 220rpm for 2h, adjust pH to <2.0 with HCl, water bath at 70°C for 1h, take 1mL reaction solution and add 1.5mL methyl tert-butyl ether (MTBE) to extract, and take the supernatant for gas chromatography detection of chirality. The results show that the intermediate product 1c has been converted into the final product. The four ketoreductases were reacted according to the above method, and the conversion rate and ee value are shown in Table 14. Ketoreductases 4 and 7 have the best effect. Since the solubility of ketoreductase 7 is better than ketoreductase 4 when heterologously expressed, ketoreductase 7 is used in the following examples.

[0226] Table 14 Comparison of ketoreductase activity

[0227]

[0228] Example 10: Scale-up reaction for preparing (R)-4-propyl-dihydrofuran-2-one using benzyl ester substrate c

[0229] Enone reductase catalytic reaction system: Enone reductase 1-63 14mg / mL, benzyl ester substrate c 10mg / mL, isopropanol 20% (v / v), 0.2M PBS buffer (pH7.0) 25mL, NADP+5mM, glucose 192mM, glucose dehydrogenase 6mg / mL, add water to prepare 100mL reaction solution. The above reaction solution was placed in a 250mL round-bottom flask, adjusted to pH7.5, and stirred at 30℃ for 1h. Take 1mL of the reaction solution and add it to 1mL of ethyl acetate. After mixing, centrifuge at 12000rpm for 5min, and take the supernatant for gas chromatography analysis. The results showed that the substrate conversion rate was 100%.

[0230] Ketoreductase catalytic reaction system: Take the first step reaction solution, add glucose 192mM, NAD+5mM, ketoreductase 7 20mg / mL, glucose dehydrogenase 6mg / mL, adjust pH to 7.0, stir at 30℃ for 2h, and monitor the disappearance of intermediate product 1c by gas chromatography. Adjust pH to <2.0 with HCl, and place in a 70℃ water bath for 1h. Take 1mL of reaction solution and add 1.5mL MTBE for extraction, and take the supernatant for gas chromatography detection of chirality. The conversion rate of intermediate product 1c is 100%, and the R configuration ee value of the final product is 94.98%.

[0231] Example 11: One-pot preparation of (R)-4-propyl-dihydrofuran-2-one using benzyl ester substrate c

[0232] The one-pot process for preparing (R)-4-propyl-dihydrofuran-2-one is shown in Fig. 9 In order to make the pH of the reaction system more stable and more conducive to industrial production, the coenzyme circulation system was adjusted from glucose dehydrogenase / glucose to isopropanol dehydrogenase / isopropanol, and the R-configuration ketone reductase 1 mutant with an ee value greater than 85% in Example 6 that reacted with the benzyl ester substrate c was selected.

[0233] One-pot catalytic reaction system: 20 mg / mL of enoketone reductase, 20 mg / mL of ketoreductase 7, 5 mg / mL of benzyl ester substrate c, 20% (v / v) of isopropanol, 1.25 mL of 0.2 M PBS buffer (pH 7.0), 5 mM NADP+, 6 mg / mL of isopropanol dehydrogenase, and water was added to prepare 5 mL of reaction solution. The above reaction solution was placed in a 50 mL centrifuge tube, adjusted to pH 7.5, reacted at 30°C, 220 rpm for 30 min, adjusted to pH < 2.0 with HCl, and placed in a 70°C water bath for 1 h. 1 mL of the reaction solution was extracted with 1.5 mL of MTBE, and the supernatant was used for gas chromatography to detect chirality. The results are shown in Table 15. The ee values ​​of the R configuration of the one-pot reaction of the selected mutants with the benzyl ester substrate c were all above 85%, and except for 1-49 and 1-55, the ee values ​​were all higher than 90%; among them, the conversion rate of mutant 1-63 was 100%, and the ee value reached 95.20%.

[0234] Table 15 One-pot reaction results of different ketone reductase 1 mutants on benzyl ester substrate c

[0235]

[0236] Example 12: One-pot feed reaction of benzyl ester substrate c

[0237] 40mg / mL of enone reductase 1-63, 20mg / mL of ketoreductase 7, 8% (v / v) of isopropanol, 5mL of 0.2M PBS buffer (pH7.0), 5mM NADP+, and 6mg / mL of isopropanol dehydrogenase were prepared into a 20mL reaction solution, placed in a 50mL centrifuge tube, and adjusted to pH7.0. 0.4mL (about 400mg) of benzyl ester substrate c was mixed with 0.4mL of isopropanol to prepare a substrate solution, which was added to the reaction solution at a flow rate of 0.5mL / h, and reacted at room temperature and 220rpm. After the substrate was added dropwise, 1mL of the reaction solution was taken, the pH was adjusted to <2.0 with HCl, and the reaction was carried out in a 70℃ water bath for 30min. 1mL of ethyl acetate was added to extract the supernatant, and the supernatant was used for gas chromatography to detect chirality. The results showed that the conversion rate of the substrate was 89.99%, and the R configuration ee value of the product was 92.54%.

[0238] The sequences of the present invention are as follows:

[0239] SEQ ID NO: 1 (amino acid sequence of ketone reductase 1)

[0240] MPFVKDFEPQALGDTNLFKPIKIGNTQPAHRAVMPPLTRMRATHPGNIPNKEWAAVYYGQRAQRPGTMIITEGTFISPQAGGYDNAPGIWSDEQVAEWKNIFLAIHDCQSFAWVQLWSLGWASFPDVLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNKRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPLLTEGEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0241] SEQ ID NO:2 (Nucleotide sequence of enone reductase 1)

[0242]

[0243] SEQ ID NO: 3 (amino acid sequence of ketone reductase 2 from Saccharomyces cerevisiae S288C)

[0244] MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTK IFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLN QFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTE GEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN

[0245] SEQ ID NO: 4 (nucleotide sequence of ketone reductase 2 from Saccharomyces cerevisiae S288C)

[0246]

[0247] SEQ ID NO: 5 (amino acid sequence of ketone reductase 3 from Chroococcidiopsis thermalis)

[0248] MNTNIDLFSPVRLGRYELPNRMVMAPLTRNRAGEGNVPRELNAEYYAQRVSAGLIITEQVSPQGLGYPFTPGIHSQEQVEGWRLVTKAVHDRGGKIFLQLWHVGRISHPDLQVDGALPVAPSAIAPSEGMAATYEGEKPYVTPRALETAEPGIVEQYRQGAKNALAAGFDGVEIHSANGYLL DQFLHDGSNHRTDEYGGSIENRARLLMEVTEAVVSVWGADRVGVRLSPSGTFGSVYDSDLKALFTYVVDALNQFELAYLHLVEPRVAGNETVENPTSELSSKYFRPIYKGTLISAGGYDRESGNAVLASGDADLVAYGRLFISNPDLPQRFALNAQLNPYDRSSFYGGDKRGYTDYPSLELQAAG

[0249] SEQ ID NO:6 (nucleotide sequence of ketone reductase 3 from Chroococcidiopsis thermalis)

[0250]

[0251] SEQ ID NO:7 (amino acid sequence of ketoreductase 4 from Equus caballus)

[0252] MSTAGKVIKCKAAVLWEEKKPFSIEEVEVAPPKAHEVRIKMVATGICRSDDHVVSGTLVTPLPVIAGHEAAGIVESIGEGVTTVRPGDKVIPLFTPQCGKCRVCKHPEGNFCLKNDLSMPRGTMQDGTSRFTCRGKPIHHFLGTSTFSQYTVVDEISVAKIDAASPLEKVCLIGCGFSTGYGSAVKV AKVTQGSTCAVFGLGGVGLSVIMCKAAGAARIIGVDINKDKFAKAKEVGATEVNPQDYKKPIQEVLTEMSNGGVDFSFEVIGRLDTMVTALSCCQEAYGVSVIVGVPPDSQNLSMNPMLLLSGRTWKGAIFGGFKSKDSVPKLVADFMAKKFALDPLITHVLPFEKINEGFDLLRSGESIRTILTF

[0253] SEQ ID NO: 8 (nucleotide sequence of ketoreductase 4 from Equus caballus)

[0254]

[0255] SEQ ID NO:9 (amino acid sequence of ketoreductase 5 from Yokenella sp. WZY002)

[0256] MSIIKSYAAKEAGSELELYEYDAGELRPEDVEVQVDYCGICHSDLSMIDNEWGFSQYPLVAGHEVIGRVAALGSAAQEKGVKVGQRVGVGWTARSCGHCDACISGNQINCLEGAVATILNRGGFAEKLRADWQWVIPLPESIDIESAGPLLCGGITVFKPLLMHHITAT SRVGVIGIGGLGHIAIKLLHAMGCEVTAFSSNPSKEQEVLAMGADKVVNSRDPDALNALAGQFDLIINTVNVDLDWQPYFEALAYGGHFHTVGAVMKPLPVPAFTLIAGDRSISGSATGTPYELRKLMKFAGRSKVSPTTELFPMSQINEAIQHVRDGKARYRVVLQADF

[0257] SEQ ID NO: 10 (nucleotide sequence of ketoreductase 5 from Yokenella sp. WZY002)

[0258]

[0259] SEQ ID NO: 11 (amino acid sequence of ketoreductase 6 from Rauvolfia serpentina)

[0260] MPRVKLGTQGLEVSKLGFGCMGLSGDYNDALPEEQGIAVIKEAFNCGITFFDTSDIYGENGSNEELLGKALKQLPREKIQVGTKFGIHEIGFSGVKAKGTPDYVRSCCEASLKRLDVDYIDLFYIHRIDTTVPIEITMGELKKLVEEGKIKYVGLSEASPDTIRRAHA VHPVTALQIEYSLWTRDIEDEIVPLCRQLGIGIVPYSPIGRGLFAGKAIKESLPENSVLTSHPRFVGENLEKNKQIYYRIEALSQKHGCTPVQLALAWVLHQGEDVVPIPGTTKIKNLHNNVGALKVKLTKEDLKEISDAVPLDEVAGESIHEVIAVTNWKFANTPPLK

[0261] SEQ ID NO: 12 (nucleotide sequence of ketoreductase 6 from Rauvolfia serpentina)

[0262]

[0263] SEQ ID NO: 13 (amino acid sequence of ketoreductase 7 from Geobacillus sp. A8)

[0264] MRLKGKAAIVTGGASGIGRATAIRFAEEGAKVAVSDINEEGGEETVRLIREKGGEAIFVQTDVADSKQVSRLVQTAVDAFGGLHILFNNAGIGHSEVRSTDLSEEEWDRVINVNLKGVFLGIKYA VPVMKQCGGGAIVNTSSLLGIKGKKYESAYNASKAGVILLTKNAALEYGKFNIRVNAIAPGVIDTNIITPWKQDERKWPIISKANALGRIGTPEEVANAVLFLASDEASFITGATLSVDGGGLTF

[0265] SEQ ID NO: 14 (nucleotide sequence of ketoreductase 7 from Geobacillus sp. A8)

[0266] ATGCGTCTGAAAGGCAAAGCGGCGATCGTTACCGGCGGCGCGAGCGGCATCGGTCGTGCGACCGCGATCCGTTTCGCTGAAGAAGGTGCGAAAGTTGCGGTTAGCGATATCAACGAAGAAGGCGGTGAAGAAACCGTTCGTCTGATCCGTGAAAAAGGCGGTGAAGCGATCTTCGTTCAGACCGATGTTGCGGATTCTAAACAGGTTTCTCGTCTGGTTCAGACCGCGGTTGATGCGTTCGGCGGTCTGCATATCCTGTTCAACAACGCGGGTATCGGCCACAGCGAAGTTCGTTCTACCGATCTGAGCGAAGAAGAATGGGACCGTGTTATCAACGTGAACCTGAAAGGCGTTTTCCTGGGCATCAAATATGCGGTTCCGGTGATGAAACAGTGCGGCGGCGGCGCTATCGTTAACACCTCTAGCCTGCTGGGTATCAAAGGCAAAAAATACGAAAGCGCGTACAACGCGTCCAAAGCGGGTGTTATCCTGCTGACCAAAAACGCGGCGCTGGAATATGGTAAATTCAACATCCGTGTTAACGCGATCGCGCCGGGCGTTATCGACACCAACATCATCACCCCGTGGAAACAGGATGAACGTAAATGGCCGATCATCAGCAAAGCGAACGCGCTGGGCCGCATCGGCACCCCGGAAGAAGTTGCGAACGCGGTTCTGTTCCTGGCGTCTGATGAAGCGAGCTTCATCACCGGCGCGACCCTGAGCGTTGATGGTGGTGGCCTGACCTTCTAA

[0267] SEQ ID NO:15 (Amino acid sequence of glucose dehydrogenase 8 derived from Bacillus subtilis 168)

[0268] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKY FVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0269] SEQ ID NO: 16 (nucleotide sequence of glucose dehydrogenase 8 from Bacillus subtilis 168)

[0270] ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCTATTACAGGAGCTGCTTCAGGGCTCGGAAAGGCGATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTATTATAGTAATAAACAAGATCCGAACGAGGTAAAAGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGATGTCACGAAAGAGGAAGATGTAAAAAATATCGTGCAAACGGCAATTAAGGAGTTCGGCACACTCGATATTATGATTAATAATGCCGGTCTTGAAAATCCTGTGCCATCTCACGAAATGCCGCTCAAGGATTGGGATAAAGTCATCGGCACGAACTTAACGGGTGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTCGTAGAAAACGATATCAAGGGAAATGTCATTAACATGTCCAGTGTGCACGAAGTGATTCCTTGGCCGTTATTTGTCCACTATGCGGCAAGTAAAGGCGGGATAAAGCTGATGACAGAAACATTAGCGTTGGAATACGCGCCGAAGGGCATTCGCGTCAATAATATTGGGCCAGGTGCGATCAACACGCCAATCAATGCTGAAAAATTCGCTGACCCTAAACAGAAAGCTGATGTAGAAAGCATGATTCCAATGGGATATATCGGCGAACCGGAGGAGATCGCCGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATCACGTTATTCGCGGACGGCGGTATGACACAATATCCTTCATTCCAGGCAGGCCGCGGTTAA

[0271] SEQ ID NO:17 (Amino acid sequence of isopropanol dehydrogenase 9 from Lactobacillus brevis KB290)

[0272] MEVVQMSNRLDGKVAIVTGGTLGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTR LGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0273] SEQ ID NO: 18 (nucleotide sequence of isopropanol dehydrogenase 9 from Lactobacillus brevis KB290)

[0274] ATGGAGGTCGTTCAAATGTCAAACCGGTTAGATGGAAAAGTAGCAATCGTTACAGGTGGTACGTTGGGTATCGGTTTAGCTATCGCCACGAAGTTCGTTGAAGAAGGGGCTAAGGTCATGATTACCGGCCGGCACAGCGATGTTGGTGAAAAAGCAGCTAAGAGTGTCGGCACTCCTGATCAGATTCAATTTTTCCAACATGATTCTTCCGATGAAGACGGCTGGACGAAATTATTCGATGCAACGGAAAAAGCCTTTGGCCCAGTTTCTACATTAGTTAATAACGCTGGGATCGCGGTTAACAAGAGTGTCGAAGAAACCACGACTGCTGAATGGCGTAAACTATTAGCCGTCAACCTTGATGGTGTCTTCTTCGGTACCCGATTAGGGATTCAACGGATGAAGAACAAAGGCTTAGGGGCTTCCATCATCAACATGTCTTCGATCGAAGGCTTTGTGGGTGATCCTAGCTTAGGGGCTTACAACGCATCTAAAGGGGCCGTACGGATTATGTCCAAGTCAGCTGCCTTAGATTGTGCCCTAAAGGACTACGATGTTCGGGTAAACACTGTTCACCCTGGCTACATCAAGACACCATTGGTTGATGACTTACCAGGGGCCGAAGAAGCGATGTCACAACGGACCAAGACGCCAATGGGCCATATCGGTGAACCTAACGATATTGCCTACATCTGTGTTTACTTGGCTTCTAACGAATCTAAATTTGCAACGGGTTCTGAATTTGTAGTTGATGGTGGTTATACCGCTCAATAA。

Claims

1. An enone reductase, characterized in that The ketene reductase has at least an amino acid residue W117 or a homologous site thereof, wherein the 117 position is numbered with reference to the amino acid residues in SEQ ID NO:

1.

2. The ketone reductase according to claim 1, wherein It is selected from the following: (1) The ketone reductase has a difference selected from the group consisting of amino acid residues W117A, W117S, W117P, W117G, W117V, W117C, W117R, W117E, W117K, W117L, W117I or W117T relative to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; preferably, it has a difference in amino acid residue W117A; More preferably, it also has one or more differences selected from the group consisting of amino acid residues L37A, T38S / L, Y83W and P296F; or, (2) the ketene reductase has a difference with respect to the amino acid sequence shown in SEQ ID NO: 5, selected from the group consisting of amino acid residues W103A, W103S, W103P, W103G, W103V, W103C, W103R, W103E, W103K, W103L, W103I or W103T; preferably, it has a difference with respect to amino acid residue W103A; More preferably, there are one or more differences selected from the group consisting of amino acid residues L27A, T28S, Y69W and E275F.

3. The ketone reductase according to claim 2, characterized in that The ketene reductase has one or more differences in amino acid residues P35C / E / A / L, M40A / F / L, I76L / V, L119I, R244A, S246T and I352L relative to the amino acid sequence shown in SEQ ID NO:

1.

4. The ketone reductase according to claim 1, wherein The ketone reductase has a difference in amino acid residues selected from the following groups relative to the amino acid sequence shown in SEQ ID NO: 1: (1)W117N, W117S, W117Q, W117P, W117G, W117D, W117V, W117C, W117E, W117Y, W117 K, W117L, W117I, W117T, W117M, W117H, W117F, W117R, P35C / L37A / T38S / Y83W / W117 V / P296F, P35C / L37A / T38S / Y83W / W117S / P296F, P35C / L37A / T38S / Y83W / W117P / P2 96F, P35C / L37A / T38S / Y83W / W117T / P296F, P35C / L37A / T38S / Y83W / W117G / P296F; ((2)W117A, Y83W / W117A, W117A / P296F, P35C / W117A, P35L / W117A, T38S / W117A, T38L / W117A, T38S / Y83W / W117A, Y 83W / W117A / P296F, T38S / Y83W / W117A / P296F, P35A / T38S / Y83W / W117A / P296F, L37A / T38S / Y83W / W117A / P296F, T3 8S / Y83W / W117A / R244A / P296F, L37A / T38S / Y83W / W117A / P296F / I352L, P35E / L37A / T38S / Y83W / W117A / P296F, P35 C / L37A / T38S / Y83W / W117A / P296F, P35C / L37A / T38S / M40A / Y83W / W117A / P296F, P35C / L37A / T38S / Y83W / W117A / L11 9I / P296F, P35C / L37A / T38S / Y83W / W117A / S246T / P296F, P35C / L37A / T38S / M40A / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / M40L / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / M40F / Y83W / W117A / L119I / P296F, P35C / L37A / T38 S / I76L / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / I76V / Y83W / W117A / L119I / P296F, P35C / L37A / T38S / Y83W / W117A / L119I / S246T / P296F, P35C / L37A / T38S / M40A / W117A / P296F or P35C / L37A / T38S / M40A / W117A / L119I / P296F; or, The ketene reductase has the following differences in amino acid residues relative to the amino acid sequence shown in SEQ ID NO: 3: W117A, Y83W / W117A, T38S / Y83W / W117A, T38S / Y83W / W117A / P296F or L37A / T38S / Y83W / W117A / P296F; or, The ketene reductase has the following differences in amino acid residues relative to the amino acid sequence shown in SEQ ID NO:5: W103A, Y69W / W103A, T28S / Y69W / W103A, T28S / Y69W / W103A / E275F, or L27A / T28S / Y69W / W103A / E275F.

5. An isolated nucleic acid, characterized in that It encodes the ketone reductase according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that: It comprises the isolated nucleic acid as claimed in claim 5; Preferably, the recombinant expression vector comprises a pET28a plasmid backbone.

7. A genetically engineered bacterium, characterized in that: It comprises the isolated nucleic acid according to claim 5 or the recombinant expression vector according to claim 6.

8. A method for producing ketone reductase, characterized in that: The genetically engineered bacteria according to claim 7 are inoculated into a culture medium suitable for their growth to obtain the enone reductase.

9. A method for producing an intermediate product 1, characterized in that The method comprises: mixing ketone reductase or a genetically engineered bacterium producing the ketone reductase with a substrate, and obtaining the intermediate product 1 after reaction; The substrate is a compound as shown in Formula 1: Wherein, R is benzyl or C1-C4 alkyl; The intermediate product 1 is a compound as shown in Formula 2: Wherein, R is benzyl or C1-C4 alkyl; Preferably, the ketene reductase is the ketene reductase according to any one of claims 1 to 4; and / or, the genetically engineered bacteria is the genetically engineered bacteria according to claim 7; More preferably, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing; Further preferably, the buffer is PBS buffer; and / or, The coenzyme is selected from any one or both of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; and / or, The coenzyme circulation system comprises glucose dehydrogenase and glucose, or, comprises isopropanol dehydrogenase and isopropanol; preferably, the glucose dehydrogenase is a glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is an isopropanol dehydrogenase derived from Lactobacillus brevis KB290; for example, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:

17.

10. A method for producing an intermediate product 2, characterized in that: The method comprises: mixing ketoreductase with an intermediate product 1, and obtaining the intermediate product 2 after reaction; The intermediate product 1 is a compound as shown in Formula 2: Wherein, R is benzyl or C1-C4 alkyl; The intermediate product 2 is a compound as shown in Formula 3: Wherein, R is benzyl or C1-C4 alkyl; Preferably, the ketoreductase is selected from ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina and Geobacillus sp. A8; for example, The amino acid sequence of the ketoreductase is selected from SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13; More preferably, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing; Further preferably, the buffer is PBS buffer; and / or, The coenzyme is selected from any one or both of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; and / or, The coenzyme circulation system comprises glucose dehydrogenase and glucose, or, comprises isopropanol dehydrogenase and isopropanol; preferably, the glucose dehydrogenase is glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is isopropanol dehydrogenase derived from Lactobacillus brevis KB290; for example, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO: 17; and / or, The intermediate product 1 is the intermediate product 1 obtained by the method according to claim 9.

11. A method for producing (R)-4-propyl-dihydrofuran-2-one as a brivaracetam intermediate, characterized in that: The method comprises: mixing ketone reductase or a genetically engineered bacterium producing the ketone reductase with a substrate to obtain an intermediate product 1 after reaction; and using the intermediate product 1 to prepare (R)-4-propyl-dihydrofuran-2-one; The substrate is a compound as shown in Formula 1: Wherein, R is benzyl or C1-C4 alkyl; The intermediate product 1 is a compound as shown in Formula 2: Wherein, R is benzyl or C1-C4 alkyl; Preferably, the ketone reductase is the ketone reductase according to any one of claims 1 to 4; and / or, the genetically engineered bacteria is the genetically engineered bacteria according to claim 7; and / or, the method for preparing (R)-4-propyl-dihydrofuran-2-one using the intermediate 1 comprises: mixing the ketone reductase with the intermediate 1, and obtaining the (R)-4-propyl-dihydrofuran-2-one after reaction; More preferably, the method for preparing (R)-4-propyl-dihydrofuran-2-one using the intermediate product 1 comprises: mixing the ketoreductase with the intermediate product 1 to obtain the intermediate product 2 after reaction; the intermediate product 2 undergoes a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one; and / or, The ketoreductase is selected from the ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina and Geobacillus sp. A8; for example, the amino acid sequence of the ketoreductase is selected from SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13; Further preferably, the enone reductase or the genetically engineered bacteria is mixed with the substrate in a buffer; and / or, coenzymes and coenzyme circulation systems are added before, during or after the enone reductase or the genetically engineered bacteria is mixed with the substrate; and / or, coenzymes and coenzyme circulation systems are added before, during or after the ketoreductase is mixed with the intermediate 1.

12. The method according to claim 11, characterized in that The buffer is PBS buffer; and / or, The coenzyme is selected from any one or both of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; and / or, The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol; Preferably, the glucose dehydrogenase is a glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is an isopropanol dehydrogenase derived from Lactobacillus brevis KB290; for example, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:

17.

13. A method for producing the intermediate (R)-4-propyl-dihydrofuran-2-one of brivaracetam, characterized in that: The method comprises: mixing ketoreductase with an intermediate product 1, and obtaining the (R)-4-propyl-dihydrofuran-2-one after reaction; The intermediate product 1 is a compound as shown in Formula 2: Wherein, R is benzyl or C1-C4 alkyl; Preferably, the method comprises: mixing the ketoreductase with the intermediate product 1 to obtain the intermediate product 2 after reaction; the intermediate product 2 undergoes a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one; and / or, The ketoreductase is selected from the ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina and Geobacillus sp. A8; for example, the amino acid sequence of the ketoreductase is selected from SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13; More preferably, the method further comprises mixing ketene reductase or a genetically engineered bacterium producing the ketene reductase with a substrate to obtain the intermediate product 1 after reaction; The substrate is a compound as shown in Formula 1: Wherein, R is benzyl or C1-C4 alkyl; More preferably, the ketene reductase is the ketene reductase according to any one of claims 1 to 4; and / or, the genetically engineered bacteria is the genetically engineered bacteria according to claim 7; and / or, The enone reductase or the genetically engineered bacteria is mixed with the substrate in a buffer; and / or, coenzymes and coenzyme circulation systems are added before, during or after the enone reductase or the genetically engineered bacteria is mixed with the substrate; and / or, coenzymes and coenzyme circulation systems are added before, during or after the ketoreductase is mixed with the intermediate 1.

14. The method according to claim 13, characterized in that The buffer is PBS buffer; and / or, The coenzyme is selected from any one or both of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; and / or, The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol; Preferably, the glucose dehydrogenase is a glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is an isopropanol dehydrogenase derived from Lactobacillus brevis KB290; for example, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:

17.

15. A method for producing the intermediate (R)-4-propyl-dihydrofuran-2-one of brivaracetam, characterized in that: The method comprises: mixing any one or two of enone reductase or genetically engineered bacteria producing the enone reductase, ketone reductase and substrate, and reacting to obtain the (R)-4-propyl-dihydrofuran-2-one; The substrate is a compound as shown in Formula 1: Wherein, R is benzyl or C1-C4 alkyl; Preferably, the method comprises: mixing any one or both of the enone reductase or the genetically engineered bacteria producing the enone reductase, the ketoreductase and the substrate, and reacting to obtain an intermediate product 2; the intermediate product 2 undergoes a ring-closing reaction to obtain the (R)-4-propyl-dihydrofuran-2-one; and / or, The enone reductase is the enone reductase according to any one of claims 1 to 4; and / or, the genetically engineered bacterium is the genetically engineered bacterium according to claim 7; and / or, the ketoreductase is selected from the ketoreductases derived from Equus caballus, Yokenella sp. WZY002, Rauvolfia serpentina and Geobacillus sp. A8; for example, the amino acid sequence of the ketoreductase is selected from SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13; More preferably, the mixing is performed in a buffer; and / or the coenzyme and the coenzyme circulation system are added before, during or after the mixing.

16. The method according to claim 15, characterized in that The buffer is PBS buffer; and / or, The coenzyme is selected from any one or both of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate; and / or, The coenzyme circulation system includes glucose dehydrogenase and glucose, or includes isopropanol dehydrogenase and isopropanol; Preferably, the glucose dehydrogenase is a glucose dehydrogenase derived from Bacillus subtilis 168; and / or, the isopropanol dehydrogenase is an isopropanol dehydrogenase derived from Lactobacillus brevis KB290; for example, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO:

17.

17. A compound, characterized in that The compound has a structure as shown in Formula 4: Wherein, R is ethyl or benzyl.

18. A method for preparing the compound according to claim 17, characterized in that: The method comprises the following steps: mixing any one of benzyl bromide or ethyl bromide with 5-hydroxy-4-n-propyl-2(5H)-furanone, and obtaining the compound after reaction; Preferably, the reaction is carried out in a mixed reaction system of dimethylformamide and potassium carbonate; More preferably, the reaction conditions satisfy one or more of the following conditions: The temperature is 25 to 35°C, for example 30°C; The time is 1.5 to 6 hours, for example 2 hours.

19. Use of the compound according to claim 17 in the production of (R)-4-propyl-dihydrofuran-2-one.

20. Use of the genetically engineered bacterium according to claim 7 in the production of ketone reductase or (R)-4-propyl-dihydrofuran-2-one, or use of the ketone reductase according to any one of claims 1 to 4 in the production of (R)-4-propyl-dihydrofuran-2-one.

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