Application of amide hydrolase in preparation of chiral amines

Abstract

The invention discloses an application of amide hydrolase in preparation of chiral amines. The invention provides a preparation method of a compound II, which comprises the following steps: in a solvent, in the presence of an enzyme, a compound I is subjected to a hydrolysis reaction as shown in the following formula to obtain the compound II, and the enzyme has an amino acid sequence as shown in SEQ ID NO: 1. The amide hydrolase with the amino acid sequence as shown in SEQ ID NO: 1 can selectively hydrolyze an L-configuration substrate to obtain an L-configuration amino acid, has a good conversion rate, and is wide in applicable substrate range. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the application of amidohydrolase in the preparation of chiral amines, in particular to the application in biocatalytic amidohydrolysis reaction. Background Art

[0002] Biocatalysis, the most important catalytic method to date, boasts advantages such as high efficiency, high selectivity, and environmental friendliness. The use of biocatalysis for the synthesis of high-value chemicals holds significant application potential and significance, particularly for the highly stereoselective synthesis of chiral chemicals. Amide hydrolases, members of the hydrolase superfamily, catalyze the hydrolysis of amides to produce the corresponding amines. This reaction can be used to prepare optically pure pharmaceuticals and chemical intermediates.

[0003] So far, there are relatively few reports on the research of amide hydrolases. In addition, the catalytic properties of the currently reported amide hydrolases are relatively poor and cannot meet the needs of being widely used. For example, the activity of the amide hydrolases from most of the microbial sources that have been found is relatively low and the thermal stability is relatively poor. For example, the amide hydrolases derived from Rhodococcus erythropolis AJ270 have been proven to be able to catalyze the hydrolysis reaction of various amide substrates to generate corresponding amine products, and have the advantages of high catalytic activity and high selectivity, and have been applied to the synthesis of various chiral non-natural amino acids and drug molecular structure skeletons. However, they show lower catalytic efficiency and enantioselectivity for some substrates. Therefore, finding amide hydrolases with different sources and excellent properties, and obtaining exogenous target proteins through heterologous expression, are important tasks in the current biotransformation of amide compounds.

[0004] Chiral amines are widely used in industries such as medicine, agriculture, food, and cosmetics. Developing efficient, atom-economical, and environmentally friendly methods for synthesizing chiral amines is of great significance. Amide hydrolases have great potential for preparing optically pure amine products. Summary of the Invention

[0005] The present invention addresses the limited availability of methods for preparing amino acids using amidohydrolases in the prior art. To address this issue, the present invention provides the use of an amidohydrolase in the preparation of chiral amines. The present invention discovers that an amidohydrolase having the amino acid sequence shown in SEQ ID NO: 1 can selectively hydrolyze L-configured substrates to produce L-configured amino acids with good conversion rates and a wide range of substrate applicability.

[0006] The present invention provides a method for preparing compound II, which comprises the following steps: in a solvent, in the presence of an enzyme, subjecting compound I to a hydrolysis reaction as shown in the following formula to obtain compound II:

[0007]

[0008] R 1 C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with one or more hydroxy groups;

[0009] R 2 is H;

[0010] or R 1 、R 2 Together with the atoms to which it is attached, it forms a 3-8 membered heterocycloalkylene group, wherein the 3-8 membered heterocycloalkylene group is optionally substituted with one or more hydroxyl groups; the heteroatom of the 3-8 membered heterocycloalkylene group is N, and the number of heteroatoms is 1, 2 or 3;

[0011] Y is a single bond or C 1-6 alkylene;

[0012] R 3 -C 1-6 Alkylene-C 6-10 Aryl or -C 1-6 Alkylene-5-10 membered heteroaryl;

[0013] The enzyme has an amino acid sequence as shown in SEQ ID NO: 1;

[0014] The carbon atom marked with "*" in compound I is a chiral carbon atom, and the chiral carbon atom is in S configuration or a mixture of R configuration and S configuration; the carbon atom marked with "*" in compound II is a chiral carbon atom, and its configuration is S configuration (i.e., L configuration).

[0015] In a certain embodiment, in the preparation method of compound II, certain groups and conditions have the following definitions, and the definitions of the groups and conditions not mentioned are as described in any embodiment of the present invention (this paragraph is hereinafter referred to as "in a certain embodiment").

[0016] In one scenario, R 1 In the C 1-6 The alkyl group is methyl or isopropyl, and the methyl and isopropyl groups are each independently optionally substituted by 1, 2 or 3 hydroxyl groups; preferably, R 1 Methyl, or isopropyl.

[0017] In one embodiment, Y is a single bond or a methylene group; for example, a single bond.

[0018] In one scenario, R 1 、R 2 Together with the atoms to which it is attached, it forms a 3-8 membered heterocycloalkylene group, wherein the 3-8 membered heterocycloalkylene group is Said Optionally substituted with 1, 2 or 3 hydroxyl groups; preferably, R 1 、R 2 Together with the atoms it is connected to form The carbon atom marked with "*" is a chiral carbon atom, and the chiral carbon atom is in S configuration, R configuration, or a mixture of R configuration and S configuration; preferably in R configuration.

[0019] In a certain embodiment, the compound I and compound II are selected from any one of the following embodiments:

[0020] Scheme 1: Compound I is Compound II is

[0021] Scheme 2: Compound I is Compound II is

[0022] Scheme 3: Compound I is Compound II is

[0023] Scheme 4: Compound I is Compound II is

[0024] Scheme 5: Compound I is Compound II is

[0025] In Schemes 1-5, the carbon atom marked with "*" is a chiral carbon atom, and the chiral carbon atom is in S configuration or a mixture of R configuration and S configuration.

[0026] The solvent is a conventional solvent for such reactions in the art. Preferably, the solvent is a buffer solution, such as a phosphate buffer solution, a potassium phosphate buffer solution, or a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution. The concentration of the dipotassium hydrogen phosphate solution may be 0.06 mol / L, and the concentration of the potassium dihydrogen phosphate solution may be 0.04 mol / L.

[0027] In one embodiment, the pH of the solvent is 7.0 to 8.0, such as 7.4 to 7.6, and further such as 7.5.

[0028] In one embodiment, the mass volume ratio of the compound I to the solvent is (1-50) g / L, for example (1-10) g / L, and another example is 4 g / L.

[0029] In one embodiment, the reaction system of the hydrolysis reaction further includes a co-solvent. Preferably, the co-solvent is a sulfone solvent or an alcohol solvent. The sulfone solvent may be dimethyl sulfoxide, and the alcohol solvent may be methanol. More preferably, the co-solvent is a sulfone solvent.

[0030] In one embodiment, the mass volume ratio of the compound I to the co-solvent is (100-1000) g / L, for example, 500 g / L.

[0031] In one embodiment, the enzymes are present in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder, either alone or together, preferably in the form of solid enzyme powder.

[0032] In the present invention, the whole-cell enzyme is produced by cells (e.g., prokaryotes) that contain the gene encoding the enzyme. Whole-cell catalysis refers to the use of intact biological organisms (i.e., cells, tissues, or even individuals) for catalytic transformations. Essentially, it utilizes enzymes within the cell for catalysis. This method is a biocatalytic technology between fermentation and free enzyme catalysis.

[0033] In the present invention, the homogenized enzyme solution is an enzyme solution obtained by resuspending wet bacteria with a resuspension solution and breaking the cells, and the wet bacteria is a precipitate obtained by solid-liquid separation (i.e., discarding the supernatant after centrifugation and taking the precipitate) of the culture solution of the prokaryotic cells.

[0034] In the present invention, cells can be disrupted by any conventional method in the art, including but not limited to high-pressure disruption, ultrasonic disruption, osmotic shock disruption, repeated freezing and thawing, lysozyme treatment or cell lysis solution treatment.

[0035] In the present invention, the crude enzyme solution is the enzyme solution obtained by removing impurities from the homogenized enzyme solution (ie, taking the supernatant after centrifugation and discarding the precipitate).

[0036] In the present invention, the solid enzyme powder is a powder obtained by drying the crude enzyme solution, and the drying can be freeze-drying.

[0037] In one embodiment, the mass ratio of the enzyme to the compound I is (1-5):1, for example (1-3):1, and further for example 2:1.

[0038] In one embodiment, the temperature of the hydrolysis reaction is 20°C-40°C, such as 28°C-32°C, and another example is 30°C.

[0039] In one embodiment, the reaction system of the hydrolysis reaction consists of the solvent, cosolvent, enzyme and compound I.

[0040] In one embodiment, the hydrolysis reaction comprises the following steps:

[0041] (I) mixing the compound I with the cosolvent to obtain a mixture;

[0042] (II) reacting the mixture in step (I) with the solvent containing the enzyme to obtain compound II.

[0043] The progress of the hydrolysis reaction is detected using conventional monitoring methods for such reactions in the art (e.g., TLC or LC-MS). The hydrolysis reaction is terminated when Compound I disappears or no longer reacts. The hydroxylation reaction can last for 10-20 hours, for example, 18 hours.

[0044] The present invention provides a method for preparing compound ID, which comprises the following steps: subjecting compound I to a hydrolysis reaction as shown in the following formula in a solvent in the presence of an enzyme to obtain compound ID:

[0045]

[0046] The carbon atom marked with "*" in compound I is a chiral carbon atom, and the chiral carbon atom is a mixture of R configuration and S configuration; the carbon atom marked with "*" in compound ID is a chiral carbon atom, and its configuration is R configuration; the R 1 、R 2 、R 3 , the definition of Y, the reaction conditions and operations are as described in any one of the preparation methods of compound II.

[0047] In one embodiment, the compound ID is selected from any of the following:

[0048] Scheme 1: Compound ID is

[0049] Scheme 2: Compound ID is

[0050] Scheme 3: Compound ID is

[0051] Scheme 4: Compound ID is

[0052] Scheme 5: Compound ID is

[0053] The present invention provides a method for preparing compound III, which comprises the following steps: in a solvent, in the presence of an enzyme, subjecting compound I to a hydrolysis reaction as shown in the following formula to obtain compound III:

[0054]

[0055] The carbon atom marked with "*" in compound I is a chiral carbon atom, and the chiral carbon atom is in S configuration or a mixture of R configuration and S configuration; the R 1 、R 2 、R 3 , the definition of Y, the reaction conditions and operations are as described in any one of the preparation methods of compound II.

[0056] In a certain embodiment, the compound III is

[0057] The present invention provides a composition comprising the compound II, and the composition is prepared by the preparation method of the compound II.

[0058] The present invention provides an isolated nucleic acid encoding the enzyme as described above; the nucleic acid has a sequence as shown in SEQ ID NO: 2.

[0059] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid described above.

[0060] The present invention also provides a transformant, wherein the transformant comprises the recombinant expression vector described above.

[0061] The present invention also provides a use of an enzyme comprising an amino acid sequence as shown in SEQ ID NO: 1 in preparing Compound II;

[0062] The compound II is as described in any one of the above items.

[0063] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, or sec-butyl.

[0064] The term "heterocycloalkyl" refers to a cyclic group having a specified number of ring atoms (eg, 3 to 8 members), a specified number of heteroatoms (eg, 1, 2, or 3), and a specified type of heteroatom (N).

[0065] The term "aryl" refers to phenyl or naphthyl and the like.

[0066] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which is a monocyclic ring. Heteroaryl groups include, but are not limited to, furanyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, indolyl, and the like.

[0067] "Ylene" refers to a divalent group, such as alkylene refers to a divalent alkyl group, heterocycloalkylene refers to a divalent heterocycloalkyl group, and the alkyl and heterocycloalkyl groups are as defined above.

[0068] The "-" at the end of a group means that the group is connected to other fragments in the molecule through this site. For example, -C 1-6 Alkylene-C 6-10 Aryl refers to the group in which the alkylene group is attached to other moieties in the molecule.

[0069] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0070] The reagents and raw materials used in the present invention are commercially available.

[0071] The positive progress of the present invention is that: the present invention discovered that the amidohydrolase having the amino acid sequence shown in SEQ ID NO: 1 can selectively hydrolyze to obtain L-configured amino acids, successfully preparing chiral valine, proline, serine, hydroxyproline, alanine and related products with good conversion rate and a wide range of applicable substrates. DETAILED DESCRIPTION

[0072] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0073] The amidohydrolase of the present invention is derived from Arthrobacter sp. YN.

[0074] SEQ ID NO: 1, wild-type amidohydrolase

[0075] MSTTPESTPLVIRNANVLDVAAGTYSTADVVSVDGKFSSVEPNAQVPSGARVIDGTGKFVIPGLIDAHVHVVASSADFRSLTFTPPSYVYAQTARIMGAMLRRGFTTVRDLSGADFGLAMAQEEGLLEGPRIHFCGHALSQTGGHGDMRLPGEDHDPNSRGCCGIGRVADGVDAVRAAAARDEIRKGAHHIKIMASGGVSSPTDRI DSTQYSMEEMRAAVEEAQAANRYVAAHAYTARAINRALEAGVRSIEHGNLLDDESLKLFLEKDAFLVPTLVTYWALKEEGKEFGLTEEMWGKVDSVLTSGLEAIARAHEAGVKMAFGSDLGGMHRHQNEQFRLLGKVQPAIDAIRSATTTAAELLEREGEIGVIAPGADADLLVLDADPVADIAVLADISEHLEYLVQNGRIIH

[0076] In the following examples, the derivatization method and detection conditions for the product amino acids are as follows: 20 μl of the reaction solution was mixed with 200 μl of 0.75 M boric acid solution (pH 7.5), 400 μl of FMOC-Cl-acetone solution (2 mg / ml FMOC-Cl in acetone) was added, and the mixture was incubated at 30°C and shaken for 20 minutes. 1 mL of methanol was then added, mixed, and centrifuged. The supernatant was collected and detected by UPCC. The detection conditions were as follows: CHIPALPAK AD-H column, 250 × 4.6 mm, 5 μm; mobile phase A was CO2, and mobile phase B was 0.2% IPAM in methanol. Gradient elution conditions were as follows: 0-6.5 min, A content decreased from 90% to 50%; 6.5-7.5 min, A content was 50%; 7.9-8.2 min, A content increased from 50% to 90%. Detection wavelength was 220 nm, column temperature was 35°C, flow rate was 1.5 ml / min, and injection volume was 1 μl.

[0077] In the following examples, the UPCC analysis conditions for other product compounds were as follows: chromatographic column: CHIRALPAK AD-3, 150*4.6 mm, 3 μm column, PN: 19524; mobile phase A: CO2, mobile phase B: 0.2% MIPA dissolved in methanol, with a ratio of A:B of 4:1; isocratic elution, detection wavelength of 220 nm; column temperature of 35°C, flow rate of 3 ml / min, injection volume of 1 μL; run time of 3 min.

[0078] Conversion rate (%) = (initial substrate amount - remaining substrate amount) / initial substrate amount × 100%.

[0079] Example 1:

[0080] The amidohydrolase polypeptide shown in SEQ ID NO: 1 was produced in E. coli BL21 (DE3) under the control of the T7 promoter. The enzyme powder was prepared as follows.

[0081] 1. Preparation of Buffer

[0082] Measure 1000 mL of purified water, add 13.921 g of dipotassium phosphate trihydrate and 5.308 g of potassium dihydrogen phosphate, and stir until completely dissolved. Control the pH to 7.4-7.6 and maintain the temperature at 20-30°C. Stir until the solids are completely dissolved. This buffer solution is named K701.

[0083] 2. Production of downstream process powder (DSP)

[0084] DSP powder provides a more purified amidohydrolase preparation from cell lysate. Larger-scale fermentations (2 L of culture) of amidohydrolase for production of DSP powder can be performed as short batches followed by fed-batch production according to standard bioprocessing methods. Briefly, amidohydrolase expression is induced by adding IPTG to a final concentration of 0.1 mM. Following fermentation expression, 20 g of cells are harvested and resuspended in 0.05 mM (pH 7.0) phosphate buffer, followed by mechanical disruption by sonication. The suspension is clarified by centrifugation at 12,000 rpm for 30 minutes. The supernatant is poured into plates and pre-frozen at -80°C for 2 hours. The enzyme concentrate is then dried in a freeze dryer and packaged to produce 2-3 g of lyophilized powder.

[0085] Example 2

[0086] A method for deracemizing the compound N-CBZ-DL-proline using a downstream process powder (DSP) product to obtain the compound N-CBZ-D-proline, L-proline, and benzyl alcohol.

[0087] This example describes a method for deracemizing N-CBZ-DL-proline using lyophilized DSP amidohydrolase powder to produce N-CBZ-D-proline, L-proline, and benzyl alcohol. The prepared lyophilized amidohydrolase powder was used in a 4 mL scale reaction. The reaction system consisted of 4 g / L of the substrate compound N-CBZ-DL-proline, dimethyl sulfoxide (DMSO), K701 buffer, and 8 g / L of DSP enzyme powder protein.

[0088] The specific steps are as follows: dissolve 20 mg of the substrate compound N-CBZ-DL-proline in 40 μL of dimethyl sulfoxide (DMSO), aspirate 32 μL and place it in 4 mL of K701 buffer (containing 32 mg of protein from a powdered DSP enzyme). Dissolve 20 mg of the substrate compound N-CBZ-DL-proline in 40 μL of dimethyl sulfoxide (DMSO), aspirate 32 μL and place it in 4 mL of K701 buffer. Place both reaction bottles at 30°C and react for 18 hours. After the reaction is complete, add 100 μL of the reaction solution to 1 mL of methanol. Centrifuge at 12,000 rpm for 5 minutes. Transfer 250 μL of the supernatant to a liquid chromatography elution bottle for UPCC analysis. The catalytic activity is determined by UPCC analysis.

[0089] After 18 hours of reaction, UPCC analysis revealed that the catalytic deracemization of N-CBZ-DL-proline produced N-CBZ-D-proline (I-5), L-proline (II-5), and benzyl alcohol with a conversion rate of 48.3%. These results indicate that the amidohydrolase has a preference for L-configuration substrates and can selectively hydrolyze them to yield the corresponding L-configuration amino acids.

[0090] Example 3:

[0091] A method for converting an amide substrate, Compound I, to a related product, Compound II, using a downstream process powder (DSP) preparation.

[0092] This example describes a method for converting a substrate compound into a primary product compound using a lyophilized DSP amidohydrolase enzyme powder. A 4 mL-scale reaction was performed using the prepared lyophilized amidohydrolase enzyme powder. The reaction system consisted of 4 g / L of the amide substrate compound, dimethyl sulfoxide (DMSO), K701 buffer, and 8 g / L of DSP enzyme powder protein.

[0093] The specific steps were as follows: 20 mg of substrate compound was dissolved in 40 μL of dimethyl sulfoxide (DMSO), and 32 μL was pipetted into 4 mL of K701 buffer (containing 32 mg of protein from a powdered DSP enzyme preparation). 20 mg of substrate compound was dissolved in 40 μL of dimethyl sulfoxide (DMSO), and 32 μL was pipetted into 4 mL of K701 buffer. Both reaction flasks were incubated at 30°C for 18 hours. A negative control was performed using an enzyme preparation transformed with an expression vector lacking the amidohydrolase gene. The results are shown below.

[0094]

[0095]

[0096] After analysis, the retention times of the products were: hydroxyproline II-1: 6.78 min; alanine II-2: 6.43 min; valine II-3: 6.17 min, serine II-4: 9.13 min, proline II-5: 7.37 min, which were consistent with the retention times of the standard.

[0097] The retention time of the D configuration of compound I-1 is 2.11 min, and the retention time of the L configuration is 2.34 min; the retention time of the D configuration of I-2 is 2.22 min, and the retention time of the L configuration is 2.29 min; the retention time of the D configuration of compound I-3 is 1.98 min, and the retention time of the L configuration is 2.38 min; the retention time of the D configuration of compound I-4 is 2.39 min, and the retention time of the L configuration is 2.49 min; the retention time of the D configuration of compound I-5 is 2.11 min, and the retention time of the L configuration is 2.28 min.

[0098] After the reaction, the reaction solution was tested, and the unreacted D configuration of compounds I-1, I-2, I-3, I-4, and I-5 was detected, with retention times consistent with the standard. The benzyl alcohol product was also detected, with a retention time of 1.92 min, consistent with the standard.

[0099] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing compound II, characterized in that: It includes the following steps: In a solvent, in the presence of an enzyme, compound I is subjected to a hydrolysis reaction as shown in the following formula to obtain compound II: R 1 C 1-6 Alkyl, the C 1-6 The alkyl group is optionally substituted with one or more hydroxy groups; R 2 is H; or R 1 、R 2 Together with the atoms to which it is attached, it forms a 3-8 membered heterocycloalkylene group, wherein the 3-8 membered heterocycloalkylene group is optionally substituted with one or more hydroxyl groups; the heteroatom of the 3-8 membered heterocycloalkylene group is N, and the number of heteroatoms is 1, 2 or 3; Y is a single bond or C 1-6 alkylene; R 3 -C 1-6 Alkylene-C 6-10 Aryl or -C 1-6 Alkylene-5-10 membered heteroaryl; The enzyme has an amino acid sequence as shown in SEQ ID NO: 1; The carbon atom marked with "*" in compound I is a chiral carbon atom, and the chiral carbon atom is in S configuration or a mixture of R and S configurations; the carbon atom marked with "*" in compound II is a chiral carbon atom, and its configuration is S configuration.

2. The method for preparing compound II according to claim 1, wherein It meets one or more of the following conditions: (1)R 1 In the C 1-6 The alkyl group is methyl or isopropyl, and the methyl and isopropyl groups are each independently optionally substituted by 1, 2 or 3 hydroxyl groups; preferably, R 1 Methyl, or isopropyl; (2) Y is a single bond or a methylene group; for example, a single bond; (3)R 1 、R 2 Together with the atoms to which it is attached, it forms a 3-8 membered heterocycloalkylene group, wherein the 3-8 membered heterocycloalkylene group is described Optionally substituted with 1, 2 or 3 hydroxyl groups; preferably, R 1 、R 2 Together with the atoms it is connected to form The carbon atom marked with "*" is a chiral carbon atom, which is in S configuration, R configuration, or a mixture of R and S configurations; preferably in R configuration.

3. The method for preparing compound II according to claim 1, wherein The compound I and compound II are selected from any of the following schemes: Scheme 1: Compound I is Compound II is Scheme 2: Compound I is Compound II is Scheme 3: Compound I is Compound II is Scheme 4: Compound I is Compound II is Scheme 5: Compound I is Compound II is In Schemes 1-5, the carbon atom marked with "*" is a chiral carbon atom, and the chiral carbon atom is in S configuration or a mixture of R and S configurations.

4. The method for preparing compound II according to claim 1, wherein It meets one or more of the following conditions: (1) The solvent is a buffer solution, such as a phosphate buffer solution, a potassium phosphate buffer solution, or a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, wherein the concentration of the dipotassium hydrogen phosphate solution may be 0.06 mol / L, and the concentration of the potassium dihydrogen phosphate solution may be 0.04 mol / L; (2) The pH of the solvent is 7.0 to 8.0, for example, 7.4 to 7.6, and for example, 7.5; (3) The mass-to-volume ratio of the compound I to the solvent is (1-50) g / L, for example (1-10) g / L, and another example is 4 g / L; (4) The reaction system of the hydrolysis reaction further includes a co-solvent. Preferably, the co-solvent is a sulfone solvent or an alcohol solvent. The sulfone solvent may be dimethyl sulfoxide, and the alcohol solvent may be methanol. More preferably, the co-solvent is a sulfone solvent. (5) The mass volume ratio of the compound I to the cosolvent is (100-1000) g / L, for example, 500 g / L; (6) The enzyme is present alone or in combination in the form of whole-cell enzyme, homogenized enzyme solution, crude enzyme solution or solid enzyme powder, preferably in the form of solid enzyme powder; (7) The mass ratio of the enzyme to the compound I is (1-5):1, for example (1-3):1, and further for example 2:1; (8) The temperature of the hydrolysis reaction is 20°C-40°C, for example 28°C-32°C, and for example 30°C.

5. The method for preparing compound II according to claim 4, wherein: The reaction system of the hydrolysis reaction consists of the solvent, cosolvent, enzyme and compound I.

6. The method for preparing compound II according to claim 5, wherein: The preparation method of the compound II comprises the following steps: (I) mixing the compound I with the cosolvent to obtain a mixture; (II) reacting the mixture in step (I) with the solvent containing the enzyme to obtain compound II.

7. A method for preparing compound ID or compound III, characterized in that: The preparation method of the compound ID comprises the following steps: in a solvent, in the presence of an enzyme, subjecting the compound I to a hydrolysis reaction as shown in the following formula to obtain the compound ID: The carbon atom marked with "*" in compound I is a chiral carbon atom, which is a mixture of R configuration and S configuration; the carbon atom marked with "*" in compound ID is a chiral carbon atom, which is R configuration; the R 1 、R 2 、R 3 , Y, the definitions of compound I and the conditions and operations of the reaction are as described in any one of claims 1-6; The preparation method of the compound III comprises the following steps: in a solvent, in the presence of an enzyme, subjecting the compound I to a hydrolysis reaction as shown in the following formula to obtain the compound III: The carbon atoms marked with "*" in Compound I are chiral carbon atoms, and the chiral carbon atoms are in S configuration or a mixture of R and S configurations; the R 1 、R 2 、R 3 , Y, the definitions of compound I and the conditions and operations of the reaction are as described in any one of claims 1-6; Preferably, the compound ID is selected from any one of the following schemes: And / or, the compound III is 8. A composition comprising the compound II according to any one of claims 1 to 6, wherein the composition is prepared by the method for preparing the compound II according to any one of claims 1 to 6.

9. An isolated nucleic acid or a recombinant expression vector comprising the same or a transformant comprising the recombinant vector, wherein the nucleic acid encodes the enzyme according to claim 1; the nucleic acid has the sequence shown in SEQ ID NO:

2.

10. Use of an enzyme comprising the amino acid sequence shown in SEQ ID NO: 1 in the preparation of compound II; The compound II is as described in any one of claims 1 to 6.

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