Method for separating chiral acid / alcohol through enzymatic esterification-hydrolysis cascade

Abstract

The invention belongs to the technical field of bioengineering and food, and discloses an enzymatic esterification-hydrolysis cascade resolution method for chiral acid / alcohol, which comprises the following steps: (1) adding a soluble salt, a hydrophilic solvent and a hydrophobic solvent into a lipase solution to construct a three-liquid-phase system; (2) adding an acid or alcohol mixed spinning type chiral substance and organic acid or organic alcohol with the carbon chain length of C1-C8 into the three-liquid-phase system in the step (1), carrying out esterification reaction under a stirring condition, and centrifuging after the reaction is finished, so as to obtain an upper phase containing a product ester; and (3) reconstructing a three-liquid-phase system by using the upper phase in the step (2), lipase liquid, soluble salt and a hydrophilic solvent, carrying out hydrolysis reaction under a stirring condition, and finally obtaining a high-optical-purity product from the middle phase. The method is simple in process, the reaction efficiency and the optical purity of the product can be improved, and the problems that a traditional system product is low in optical purity, a two-step cascade reaction system is poor in compatibility, and the product and a substrate are difficult to separate are solved.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering and food technology, and relates to the separation of chiral substances and the application of enzymes, in particular to a method for enzymatic esterification-hydrolysis cascade resolution of chiral acids / alcohols. Background Art

[0002] Chiral acids / alcohols are carboxylic acids or alcohol compounds containing chiral centers, and there are significant differences in their enantiomers in terms of biological activity, pharmacological effects, and toxicity. With the rapid growth of the demand for chiral acid / alcohol drugs, the development of single enantiomer drugs has become an important research direction in medicinal chemistry. However, the separation and purification technology of chiral acids / alcohols remains the key bottleneck restricting their industrial application. Currently, the main methods for the resolution of chiral acids / alcohols include crystallization, chromatography, chemical resolution, and enzymatic resolution. Among them, enzymatic resolution is considered to be one of the most promising technologies due to its mild reaction conditions, high stereoselectivity, and environmental friendliness. However, traditional enzyme-catalyzed systems face many challenges in practical applications: the chiral product and the enantiomeric by-product have similar physicochemical properties, making it difficult to separate them efficiently; the requirement for the optical purity of the product is extremely high (usually reaching more than 99%), while the ee value of a single-step enzyme-catalyzed reaction is usually lower than 90%; in addition, traditional enzyme-catalyzed systems also have problems such as many side reactions and low conversion rates, making it difficult to meet the industrial requirements. Summary of the Invention

[0003] The object of the present invention is to provide a method for enzymatic esterification-hydrolysis cascade resolution of high-purity chiral acids / alcohols based on a three-liquid-phase system, aiming at the problems in the process of traditional enzyme-catalyzed resolution of chiral substances, such as complex multi-step purification, many side reactions, the ee value usually being lower than 90%, and low conversion rate.

[0004] In order to construct a more efficient cascade reaction system and realize the coordinated progress of esterification and hydrolysis reactions, the key of the present invention lies in finding a substrate that can support both esterification and hydrolysis reactions. Through in-depth research, we found that the nature of the hydroxy donor has a decisive influence on this process, especially medium and short-chain hydroxy donor substrates can meet the requirements of both esterification and hydrolysis reactions. Based on this discovery, we successfully constructed a three-liquid-phase esterification-hydrolysis cascade reaction system. This system can efficiently prepare chiral acids / alcohols with high optical purity through simple operations.

[0005] The reaction formula of the chiral acids / alcohols of the present invention is as follows:

[0006]

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A method for enzymatic esterification-hydrolysis cascade resolution of chiral acids / alcohols, comprising the following steps:

[0009] (1) Add soluble salts, hydrophilic solvents and hydrophobic solvents to the lipase enzyme solution to construct a three-liquid-phase system;

[0010] (2) Add acid or alcohol racemic chiral substances and organic acids or organic alcohols to the three-liquid-phase system in step (1), and carry out an esterification reaction under stirring conditions. After centrifugation at the end of the reaction, obtain the upper phase containing the product ester; the carbon chain lengths of the organic acids and organic alcohols are C1-C8;

[0011] (3) Reconstruct a three-liquid-phase system with the upper phase in step (2), lipase enzyme solution, soluble salts and hydrophilic solvents, and carry out a hydrolysis reaction under stirring conditions, and finally obtain a product with high optical purity from the middle phase.

[0012] Preferably, the carbon chain lengths of the organic acids and organic alcohols in step (2) are C2-C6.

[0013] Preferably, the organic alcohol in step (2) is one or more of ethanol, propanol, n-butanol, n-pentanol, n-hexanol, and the organic acid is one or more of acetic acid, propionic acid, n-butyric acid, n-valeric acid, n-hexanoic acid.

[0014] Preferably, the soluble salts in steps (1) and (3) are one or more of sodium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, sodium citrate, magnesium sulfate, potassium dihydrogen phosphate; the hydrophilic solvents are one or more of PEG200, PEG400, PEG600, ethanol, polypropylene glycol, [BMIM]BF 4 、[BMIM]PF 6 and one or more of them;

[0015] Preferably, the hydrophobic solvents in step (1) are one or more of isooctane, n-hexane, cyclohexane, isopropyl ether, ethyl acetate.

[0016] Preferably, the mass ratios of the soluble salts, extremely hydrophilic solvents and hydrophobic solvents to the enzyme solution in the three-liquid-phase system in step (1) are 0.2-0.8:0.2-0.8:0.1-4.0 respectively; the mass ratios of the soluble salts, extremely hydrophilic solvents and hydrophobic solvents to the enzyme solution in the three-liquid-phase system in step (3) are 0.2-0.8:0.2-0.8:0.2-4.0; the enzyme solution concentration is 1-1000 U / ml.

[0017] Preferably, the reaction conditions for steps (2) and (3) are: temperature 30-40 °C, reaction time 0.5 h-18 h.

[0018] Preferably, the lipase in step (1) is Lipase AY30 or CALB.

[0019] Preferably, the molar ratio of the organic acid or organic alcohol described in step (2) to the racemic chiral substance is 0.5 to 5; the molar volume ratio of the racemic chiral substance to the hydrophobic solvent is 20 - 150 mmol / L.

[0020] Preferably, the racemic chiral substance is a racemic chiral substance, and the racemic chiral substance is selected from any one or more of ibuprofen, mandelic acid, naproxen, 1-(4-methoxyphenyl)ethanol, 2-phenylpropionic acid, and 1-(4-methylphenyl)-1-ethanol.

[0021] The esterification-hydrolysis cascade resolution of chiral acids / alcohols is a dynamic kinetic resolution strategy based on the stereoselectivity of lipase. Its core mechanism lies in using the configuration-specific recognition ability of enzyme catalysts to achieve efficient enantiomeric separation. Specifically, in the esterification reaction stage, lipase preferentially catalyzes the esterification reaction of a chiral acid or alcohol with a specific configuration (such as R-type or S-type) in the racemic chiral mixture with a hydroxyl donor through the stereoselective binding effect of its active site, generating ester derivatives with a single enantiomeric excess. The unreacted substrate of the opposite configuration remains in the reaction system due to differences in steric hindrance or affinity, completing the first-stage esterification resolution. On this basis, through simple treatment, the generated configuration-specific ester is transferred to the hydrolysis reaction system, and lipase further selectively catalyzes the cleavage of the ester bond, preferentially hydrolyzing the target enantiomeric ester to generate chiral acid / alcohol products with high optical purity while retaining the stability of the non-target configuration ester, completing the second-stage hydrolysis resolution. Through the two-stage selective catalytic process, not only the efficient resolution of chiral substrates is achieved, but also the optical purity of the products is significantly improved, and there is no need to introduce additional chiral reagents or complex separation steps, providing an important technical route for the green synthesis of chiral drug intermediates.

[0022] The present invention realizes the efficient integration, separation, and purification of esterification-hydrolysis reactions and greatly improves the conversion rate by constructing a three-liquid-phase system of hydrophobic solvent / hydrophilic polymer / brine phase. This system has the following beneficial effects:

[0023] (1) In terms of separation and purification: The three-liquid-phase system can simultaneously achieve the directional distribution and separation of products, substrates, and enzymes, breaking through the limitations of traditional multi-step purification processes. Through the selective distribution of each component in different phases, the separation and purification process is significantly simplified, and the process efficiency is improved.

[0024] (2) In terms of reaction integration: This system realizes the continuous progress of esterification and hydrolysis reactions, solving the problems of system switching and intermediate separation caused by differences in reaction systems in traditional processes. This integrated design not only improves the continuity of the process but also effectively improves the optical purity of chiral substances.

[0025] (3) In terms of conversion rate: By selecting medium- and short-chain hydroxyl donors for the esterification reaction, the resulting product ester is more conducive to subsequent hydrolysis reactions, which can greatly improve the conversion rate. Description of the Drawings

[0026] Figure 1 It is the HPLC chart of the esterification-hydrolysis cascade reaction of 1-(4-methoxyphenyl)ethanol and n-hexanoic acid. (a), (b), (c), and (d) respectively refer to the upper phase after the esterification reaction, the middle phase after the esterification reaction, the upper phase after the hydrolysis reaction, and the middle phase after the hydrolysis reaction; A, B, C, and D are (R)-1-(4-methoxyphenyl)ethyl hexanoate, (S)-1-(4-methoxyphenyl)ethyl hexanoate, (R)-1-(4-methoxyphenyl)ethanol, and (S)-1-(4-methoxyphenyl)ethanol respectively.

[0027] Figure 2 It is the HPLC chart of the esterification-hydrolysis cascade reaction of naproxen and n-butanol. (a), (b), (c), and (d) respectively refer to the upper phase after the esterification reaction, the middle phase after the esterification reaction, the upper phase after the hydrolysis reaction, and the middle phase after the hydrolysis reaction; E, F, G, and H are (R)-naproxen butyl ester, (S)-naproxen butyl ester, (R)-naproxen, and (S)-naproxen respectively. Detailed Embodiments

[0028] The present invention will be further described in detail below with reference to specific embodiments. However, the embodiments of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0029] In this embodiment, the CALB (Candida antarctica lipase B) lipase used was purchased from Novozyme, and the lipase AY30 (Candida rugosa lipases) was purchased from Amano Corporation of Japan.

[0030] Example 1

[0031] Weigh AY30 lipase and dissolve it in deionized water to prepare an enzyme solution of 100 U / mL. Take 6.6 g of the enzyme solution and mix it with 1.6 g of anhydrous sodium sulfate. Then, add 1.8 g of PEG400 and 3.4 mL of isooctane in sequence to construct a three-liquid-phase system. Add 90 mM ibuprofen (the molar volume ratio of ibuprofen to isooctane is 90 mmol / L) and 180 mM methanol (the molar ratio of methanol to ibuprofen is 2) as acyl donors, and react with shaking at 37 °C and 200 rpm for 12 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, the esterification product is mainly distributed in the upper phase. Analyze each phase by high-performance liquid chromatography (HPLC), and the measured conversion rate is 29.74%, and the enantiomeric excess value (ee p) was 90.97%. The upper phase was washed with 1M NaHCO 3 and washed with water three times. The upper phase containing the purified product ester (0.3 g) was reconstituted with 0.99 g of AY30 enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400 to form a three-liquid-phase system, and the hydrolysis reaction was carried out under the same conditions. HPLC analysis showed that the hydrolysis conversion rate reached 46.75%, and the ee p increased from 90.97% after the esterification reaction to 99.47%.

[0032] Control Example 1

[0033] Weighed AY30 lipase was dissolved in deionized water to prepare a 100 U / mL enzyme solution. After mixing 6.6 g of the enzyme solution with 1.6 g of anhydrous sodium sulfate, 1.8 g of PEG400 and 3.4 mL of isooctane were added in sequence to construct a three-liquid-phase system. 90 mM ibuprofen and 180 mM lauryl alcohol were added as acyl donors, and the reaction was carried out with shaking at 37 °C and 200 rpm for 12 h. After the reaction solution was centrifuged at 6000 rpm for 3 min, the esterification product was mainly distributed in the upper phase. Each phase was analyzed by HPLC, and the conversion rate was measured to be 49.00%, and the ee p was 91.53%. The upper phase was washed with 1M NaHCO 3 and washed with water three times. The upper phase containing the purified product ester (0.3 g) was reconstituted with 0.99 g of AY30 enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400 to form a three-liquid-phase system, and the hydrolysis reaction was carried out under the same conditions. HPLC analysis showed that almost no hydrolysis occurred (conversion rate <1%), so the whole reaction could not proceed.

[0034] Example 2

[0035] CALB lipase was dissolved in deionized water to prepare a 5 U / mL enzyme solution. 6.6 g of the enzyme solution was mixed with 1.6 g of anhydrous sodium sulfate, and 1.8 g of PEG400 and 3.4 mL of isooctane were added in sequence to construct a three-liquid-phase system. 90 mM 1-(4-methoxyphenyl)ethanol and 180 mM n-butyric acid were added as acyl donors, and the reaction was carried out with shaking at 37 °C and 200 rpm for 2 h. After the reaction solution was centrifuged at 6000 rpm for 3 min, the esterification product was mainly distributed in the upper phase, while the unreacted substrate was mainly enriched in the middle phase. Each phase was analyzed by HPLC, and the conversion rate was measured to be 16.69%, and the ee p was 92.49%. The upper phase was extracted with the reconstituted middle phase. The upper phase containing the purified product ester (0.3 g) was reconstituted with 0.99 g of the enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400 to form a three-liquid-phase system, and the hydrolysis reaction was carried out under the same conditions. HPLC analysis showed that the hydrolysis conversion rate reached 89.22%, and the ee p increased from 92.49% after the esterification reaction to 99.64%.

[0036] The reactions were carried out using different hydroxyl donors, and the results are shown in Table 1 in detail.

[0037] Table 1 Reaction effects of different hydroxyl donors on the esterification-hydrolysis cascade resolution of chiral acids / alcohols

[0038]

[0039] Control Example 2

[0040] CALB lipase was dissolved in deionized water to prepare an enzyme solution of 5 U / mL. 6.6 g of the enzyme solution was mixed with 1.6 g of anhydrous sodium sulfate, and 1.8 g of PEG400 and 3.4 mL of isooctane were added sequentially to construct a three-liquid-phase system. 90 mM of 1-(4-methoxyphenyl)ethanol and 180 mM of lauric acid were added as acyl donors, and the reaction was carried out with shaking at 37 °C and 200 rpm for 2 h. After the reaction solution was centrifuged at 6000 rpm for 3 min, the esterification product was mainly distributed in the upper phase, while the unreacted substrate was mainly enriched in the middle phase. Each phase was analyzed by HPLC, and the conversion rate was measured to be 30.31%, and the ee p was 89.29%. The upper phase was extracted with the reconstituted middle phase. The upper phase (0.3 g) containing the purified product ester was reconstituted with 0.99 g of the enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400 to form a three-liquid-phase system, and the hydrolysis reaction was carried out under the same conditions. HPLC analysis showed that the hydrolysis conversion rate was only 33.72%, and the ee p was increased from 89.29% after the esterification reaction to 98.57%.

[0041] Example 3

[0042] CALB lipase was dissolved in deionized water to prepare an enzyme solution of 5 U / mL. 6.6 g of the enzyme solution was mixed with 1.6 g of anhydrous sodium sulfate, and 1.8 g of PEG400 and 3.4 mL of isooctane were added sequentially to construct a three-liquid-phase system. 90 mM of 1-(4-methoxyphenyl)ethanol and 180 mM of n-hexanoic acid were added as acyl donors, and the reaction was carried out with shaking at 37 °C and 200 rpm for 1 h. After the reaction solution was centrifuged at 6000 rpm for 3 min, the esterification product was mainly distributed in the upper phase, while the unreacted substrate was mainly enriched in the middle phase. Each phase was analyzed by high performance liquid chromatography (HPLC), and the conversion rate was measured to be 24.34%, and the enantiomeric excess value (ee p ) of the product was 92.21%. The upper phase was extracted with the reconstituted middle phase. The upper phase (0.3 g) containing the purified product ester was reconstituted with 0.81 g of the enzyme solution, 0.39 g of ammonium sulfate, and 0.3 g of PEG600 to form a three-liquid-phase system, and the hydrolysis reaction was carried out under the same conditions. HPLC analysis showed that the hydrolysis conversion rate reached 94.48%, and the ee p was increased from 92.21% after the esterification reaction to 99.24%.

[0043] Control Example 3

[0044] Mix CALB enzyme solution (5 U / mL, 6.6 g) with 3.4 mL of isooctane to construct an isooctane-water two-phase system. Add 90 mM 1-(4-methoxyphenyl)ethanol and 180 mM n-hexanoic acid as acyl donors, and react with shaking at 37 °C and 200 rpm for 1 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, HPLC analysis shows that the conversion rate is 13.71%, and the ee p is only 66.94%. Research shows that in the isooctane-water two-phase system, since effective phase separation of the product and the substrate cannot be achieved (the product ester and the unreacted substrate coexist in the same phase), it is necessary to separate the product through complex extraction or column chromatography and other steps, which significantly increases the operation difficulty of the subsequent hydrolysis reaction. Therefore, the subsequent hydrolysis reaction is not carried out.

[0045] Example 4

[0046] Dissolve AY30 lipase in deionized water to prepare a 100 U / mL enzyme solution. Take 0.99 g of the enzyme solution and mix it with 0.24 g of anhydrous ammonium sulfate. Then add 0.27 g of PEG400 and 0.75 mL of isooctane in sequence to construct a three-phase system. Add 60 mM naproxen and 180 mM n-butanol as acyl donors, and react with shaking at 37 °C and 200 rpm for 6 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, the esterification product is mainly distributed in the upper phase, while the unreacted substrate is mainly enriched in the middle phase. Analyze each phase by HPLC, and the measured conversion rate is 24.08%, and the enantiomeric excess value of the product is 98.21%. Directly reconstruct a three-phase system with the upper phase (0.3 g) containing the purified product ester, 0.99 g of the enzyme solution, 0.24 g of ammonium sulfate, and 0.27 g of PEG400, and carry out the hydrolysis reaction under the same conditions. HPLC analysis shows that the hydrolysis conversion rate reaches 72.99%, and the ee p increases from 98.21% after the esterification reaction to 99.65%.

[0047] Control Example 4

[0048] Mix AY30 enzyme solution (100 U / mL, 0.99 g) with 0.75 mL of isooctane to construct an isooctane-water two-phase system. Add 60 mM naproxen and 180 mM lauryl alcohol as acyl donors, and react with shaking at 37 °C and 200 rpm for 6 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, HPLC analysis shows that the conversion rate is only 1.84%, and the ee p is 98.34%, and the esterification reaction hardly proceeds, so the subsequent hydrolysis reaction cannot be carried out.

[0049] Example 5

[0050] Weigh AY30 lipase and dissolve it in deionized water to prepare an enzyme solution of 100 U / mL. Take 6.8 g of the enzyme solution and mix it with 1.6 g of anhydrous sodium sulfate, then successively add 1.6 g of PEG400 and 5 mL of isooctane to construct a three-liquid-phase system. Add 60 mM 2-phenylpropionic acid and 120 mM n-butanol as acyl donors, and react with shaking at 37 °C and 200 rpm for 12 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, the esterification product is mainly distributed in the upper phase. Analyze each phase by HPLC, and the conversion rate is measured to be 60.63%, and the ee p is 24.24%. The upper phase is washed with 1 M NaHCO 3 and then with water. Reconstruct a three-liquid-phase system with the upper phase (0.3 g) containing the purified product ester, 0.99 g of AY30 enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400, and carry out a hydrolysis reaction under the same conditions. HPLC analysis shows that the hydrolysis conversion rate reaches 55.50%, and the ee p increases from 24.24% after the esterification reaction to 93.19%.

[0051] Example 6

[0052] Weigh CALB lipase and dissolve it in deionized water to prepare an enzyme solution of 10 U / mL. Take 6.8 g of the enzyme solution and mix it with 1.6 g of anhydrous sodium sulfate, then successively add 1.6 g of PEG400 and 5 mL of isopropyl ether to construct a three-liquid-phase system. Add 60 mM mandelic acid and 120 mM n-butanol as acyl donors, and react with shaking at 37 °C and 200 rpm for 12 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, the esterification product is mainly distributed in the upper phase. Analyze each phase by HPLC, and the conversion rate is measured to be 18.24%, and the ee p is 27.62%. The upper phase is washed with 1 M NaHCO 3 and then with water three times. Reconstruct a three-liquid-phase system with the upper phase (0.3 g) containing the purified product ester, 0.99 g of CALB enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400, and carry out a hydrolysis reaction under the same conditions for 1 h. HPLC analysis shows that the hydrolysis conversion rate reaches 79.58%, and the ee p increases from 27.62% after the esterification reaction to 76.85%.

[0053] Example 7

[0054] Weigh CALB lipase and dissolve it in deionized water to prepare an enzyme solution with a concentration of 5 U / mL. Take 6.8 g of the enzyme solution and mix it with 1.6 g of anhydrous sodium sulfate. Then, add 1.6 g of PEG400 and 5 mL of isooctane in sequence to construct a three-liquid-phase system. Add 60 mM of 1-(4-methylphenyl)-1-ethanol and 120 mM of n-butyric acid as acyl donors, and react with shaking at 37 °C and 200 rpm for 2 h. After the reaction solution is centrifuged at 6000 rpm for 3 min, the esterification product is mainly distributed in the upper phase. Analyze each phase by HPLC, and the conversion rate is measured to be 24.91%, and the ee p is 97.72%. Reconstitute the middle phase to extract the upper phase. Reconstitute a three-liquid-phase system with the upper phase (0.3 g) containing the purified product ester, 0.99 g of CALB enzyme solution, 0.24 g of sodium sulfate, and 0.27 g of PEG400, and carry out a hydrolysis reaction for 4 h under the same conditions. HPLC analysis shows that the hydrolysis conversion rate reaches 83.78%, and the ee p increases from 97.72% after the esterification reaction to 99.25%.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for enzymatic esterification-hydrolysis cascade splitting of chiral acids / alcohols, characterized in that: The following steps are involved: (1) adding a soluble salt, a hydrophilic solvent and a hydrophobic solvent to a lipase solution to construct a three-liquid phase system; (2) adding a rotatory chiral substance of an acid or alcohol and an organic acid or an organic alcohol to the three-phase system of step (1), carrying out an esterification reaction under stirring conditions, and obtaining an upper phase containing a product ester after centrifugation after the reaction is completed; the carbon chain length of the organic acid or organic alcohol is C1-C8; (3) The upper phase of step (2) is reconstructed into a three-liquid phase system with lipase solution, soluble salt and hydrophilic solvent, and a hydrolysis reaction is carried out under stirring conditions, and finally a high optical purity product is obtained from the middle phase.

2. The method according to claim 1, characterized in that The carbon chain length of the organic acid or organic alcohol in step (2) is C2-C6.

3. The method according to claim 2, characterized in that In step (2), the organic alcohol is one or more of ethanol, propanol, n-butanol, n-pentanol and n-hexanol, and the organic acid is one or more of acetic acid, propionic acid, n-butyric acid, n-pentanoic acid and n-hexanoic acid.

4. The method according to claim 3, characterized in that The soluble salt in steps (1) and (3) is one or more of sodium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, sodium citrate, magnesium sulfate, and potassium dihydrogen phosphate; the hydrophilic solvent is one or more of PEG200, PEG400, PEG600, ethanol, polypropylene glycol, [BMIM]BF4, and [BMIM]PF6.

5. The method according to claim 4, characterized in that The hydrophobic solvent in step (1) is one or more of isooctane, n-hexane, cyclohexane, isopropyl ether and ethyl acetate.

6. The method according to claim 5, characterized in that In step (1), the mass ratios of soluble salt, polar aqueous solvent and hydrophobic solvent to the enzyme solution in the three-phase liquid system are 0.2-0.8:0.2-0.8:0.1-4.0 respectively; in step (3), the mass ratios of soluble salt, polar aqueous solvent and hydrophobic solvent to the enzyme solution in the three-phase liquid system are 0.2-0.8:0.2-0.8:0.2-4.0; and the concentration of the enzyme solution is 1-1000 U / ml.

7. The method according to claim 6, characterized in that The reaction conditions of steps (2) and (3) are: temperature 30-40° C., and reaction time 0.5 h-18 h.

8. The method according to any one of claims 1 to 7, characterized in that: The lipase in step (1) is LipaseAY30 or CALB.

9. The method according to claim 8, characterized in that In step (2), the molar ratio of the organic acid or organic alcohol to the rotatory chiral substance is 0.5-5; the molar volume ratio of the rotatory chiral substance to the hydrophobic solvent is 20-150 mmol / L.

10. The method according to claim 9, characterized in that The racemic chiral substance is a racemic substance, preferably any one or more of ibuprofen, mandelic acid, naproxen, 1-(4-methoxyphenyl)ethanol, 2-phenylpropionic acid, and 1-(4-methylphenyl)-1-ethanol.

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