WO2020119280A1 - 一种酶法拆分手性物质的方法 - Google Patents

一种酶法拆分手性物质的方法 Download PDF

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WO2020119280A1
WO2020119280A1 PCT/CN2019/113139 CN2019113139W WO2020119280A1 WO 2020119280 A1 WO2020119280 A1 WO 2020119280A1 CN 2019113139 W CN2019113139 W CN 2019113139W WO 2020119280 A1 WO2020119280 A1 WO 2020119280A1
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liquid layer
product
chiral
ester
reaction
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李志刚
苏金芬
杨博
王永华
李振成
陈华勇
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South China University of Technology SCUT
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • C—CHEMISTRY; METALLURGY
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01074—Cutinase (3.1.1.74)

Definitions

  • the invention belongs to the technical field of biological engineering and food, and relates to the separation and application technology of enzymes, in particular to a method for separating chiral substances by using an enzymatic method.
  • the preparation of single configuration isomers is mainly through the method of induced crystallization or chemical resolution method to resolve the racemate.
  • the traditional process usually uses chiral resolving agent to resolve, the efficiency is low, and it needs to be refined after many times. meet the target.
  • the entire process is extremely complex and extremely costly (J. Mol. Catal. B: Enzym., 2010, 62(2): 162 ⁇ 168, Org. Biomol. Chem., 2014, 12, 6634-6642, J. Mol. Catal. B: Enzym., 2010, 65(1/4):49 ⁇ 51).
  • the enzymatic resolution has the advantages of mild conditions and high product purity, which is expected to be high.
  • a method for enzymatic resolution of chiral materials solves the problems of low efficiency, high cost, poor chiral selectivity, low recovery rate, and difficulty in industrialization of existing enzymatic chiral resolution.
  • the purpose of the present invention is to provide a high-efficiency and rapid response to the problems of high cost, difficulty in continuous production, low reaction efficiency and long reaction time caused by product inhibition in the current enzymatic resolution of chiral materials. Enzymatic method for producing chiral materials.
  • a method for enzymatic resolution of chiral substances includes the following steps:
  • the three-liquid-phase system is subjected to an enzyme-catalyzed reaction under stirring conditions. After the reaction is completed, it is allowed to stand or centrifuge until it is divided into three layers, from top to bottom to the upper liquid layer, the middle liquid layer and the lower liquid layer, and then hydrolyzed.
  • the resulting single optically chiral product is mainly enriched in the middle liquid layer or the lower liquid layer, and the upper liquid layer product is another ester or amide product containing a single optically chiral product.
  • the hydrophilic solvent in step (1) is one or both of polyethylene glycol, polypropylene glycol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol and acetone More than one species; or the hydrophilic solvent is one or more of [BMIM]Br, [BMIM]BF 4 , [EMIM]ETSO 4 and [OMIM]Cl.
  • the soluble salt in step (1) is one or two of sodium citrate, sodium chloride, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate the above.
  • the hydrophobic solvent in step (1) is one or more of n-hexane, diethyl ether, isopropyl ether, ethyl acetate, cyclohexanol, petroleum ether, isooctane, benzene and toluene.
  • the reaction conditions in step (2) are: a temperature of 30 ⁇ 45°C, and a reaction time of 20min ⁇ 4h.
  • the reaction operation is generally carried out at a temperature lower than the volatile solvent.
  • the pH of the system can be adjusted.
  • the pH value of the three-liquid system in step (1) is 5-9.
  • the concentration of lipase in step (1) is 5-2000 U/mL.
  • the mass ratio of the soluble salt, the hydrophilic solvent and the hydrophobic solvent to the enzyme liquid is 0.2-0.8, 0.2-0.8 and 0.2-4, respectively.
  • the ester or amide compound composed of the racemic chiral substance is selected from the group consisting of meso-type mandelic acid methyl ester, meso-type naproxen methyl ester, meso-type (4-methoxy-phenyl )-1-Ethanol acetate, mixed-rotation 1-(4-methoxyphenyl)ethanol acetate, mixed-rotation 6-methyl 5-heptenyl 2-alcohol acetate One or more than two species.
  • the ester or amide compound composed of the racemic chiral substance accounts for 0.1-10% of the mass of the hydrophobic solvent, preferably 1% to 5%.
  • the lipase may be derived from nature or may be produced by artificial fermentation. It can be fermentation broth, crude enzyme after simple purification, or pure enzyme after purification.
  • the invention overcomes the problems of high cost, difficulty in continuous production, low reaction efficiency and long reaction time caused by product inhibition during the current enzymatic resolution and simultaneous recovery of chiral materials, and provides a crude enzyme Method for separating lipase in liquid and using lipase to produce chiral substance.
  • the process is simplified, the separation time is short, the cost is reduced, and the economy is feasible.
  • the specific performance is that: using a three-liquid phase system, the ester or amide substrate of a single optical chiral product can be distributed in the hydrophobic phase, the lipase can be distributed in the solvent-rich phase or the salt-rich phase, and the single optical chirality after hydrolysis
  • the product is mainly concentrated in another phase, and the product can be recovered and reused by simple centrifugation or standing. This process is not only conducive to the purification and recovery of enzymes, but also improves the catalytic efficiency and greatly reduces the loss of enzymes and purification costs.
  • the method has the advantages of low energy consumption, high utilization rate of raw materials, mild reaction conditions, etc., and solves the technical problem that it is difficult to industrialize the chiral substance produced by the enzymatic resolution.
  • CALB Candidadida antarctica lipase B
  • Novozyme-51032 cutinase lipase used in this example were purchased from Novozyme
  • lipase AY30 Candida rugosa lipases was purchased from Amano, Japan.
  • CALB lipase Take an appropriate amount of CALB lipase in an Erlenmeyer flask, add 1000 times the volume of water, after mixing, take 1g of crude enzyme solution (enzyme concentration 5U/mL), 0.6g of dipotassium hydrogen phosphate, add 0.4g after mixing Polyethylene glycol 400 and 0.4g of isopropyl ether solution containing 5% mixed-spin methyl mandelic acid, filled into a stoppered flask, mix evenly, the pH of the mixed solution is 8.9, and placed at a constant temperature of 200rpm On a shaker, the reaction was controlled at 30°C for 2h.
  • lipase AY30 Take an appropriate amount of lipase AY30 in an Erlenmeyer flask, add 100 times the volume of water, mix it, take 0.975g of crude enzyme solution (enzyme concentration 100U/mL), 0.225g of sodium sulfate, mix and add 0.3g of poly Ethylene glycol 400 and 0.3g of isooctane solution containing 0.5% mixed naproxen methyl ester, filled into a stoppered flask, mixed evenly, the pH value of the mixed solution was 7.0, and placed at a constant temperature of 200rpm On a shaker, the reaction was controlled at 37°C for 4h.
  • the optical rotation and enantioselectivity E value of R-naproxide product in the system reached 92.9% and 46, respectively, while the control group was only divided into upper and lower phases, and the product was distributed in both phases.
  • the E values of enantioselectivity are only 86.5% and 18.
  • lipase AY30 in an Erlenmeyer flask, add 100 times the volume of water, mix it, take 0.975g of crude enzyme solution (enzyme concentration 200U/mL), 0.24g of sodium sulfate, add 0.285g of polymer after mixing Ethylene glycol 600 and 0.3g of an isooctane solution containing 0.5% mixed spin (4-methoxy-phenyl)-1-ethanol acetate, filled into a stoppered flask to mix evenly, the pH of the mixed solution The value is 7.0, and it is placed on a constant temperature shaker with a rotation speed of 200 rpm and controlled to react at 45°C for 20 min.
  • optical rotation and enantioselectivity E of (4-methoxy-phenyl)-1-ethanol in the system have reached the values respectively. 98.13% and 232.8, while the control group is only divided into upper and lower phases, the product is distributed in both phases, the optical rotation and enantioselectivity E of the products are only 94.13% and 69.39.
  • lipase AY30 in an Erlenmeyer flask, add 100 times the volume of water, mix it, take 0.975g of crude enzyme solution (enzyme concentration 100U/mL), 0.225g of sodium sulfate, mix and add 0.3g [ BMIm] BF 4 and 0.3g of isooctane solution containing 0.5% mixed-rotation naproxen methyl ester, filled into a stoppered flask and mixed evenly, and placed on a constant temperature shaker with a rotation speed of 200rpm, controlled at 37°C Reaction 4h. After the reaction, the centrifuge was set at 3000 rpm for 1 min, divided into three layers, followed by upper liquid layer, middle liquid layer and lower liquid layer.
  • the single optically chiral products after hydrolysis were mainly concentrated in the middle liquid layer, and the upper liquid layer was collected.
  • the product is an ester product of another single optically chiral product.
  • Lipase is mainly distributed in the lower liquid layer, and the enzyme recovery rate of the lower liquid layer can reach 98.5%.
  • the optical rotation and enantioselectivity E value of R-naproxide production in the system also reached 82.1% and 11 respectively.
  • CALB lipase Take an appropriate amount of CALB lipase in an Erlenmeyer flask, add 100 times the volume of water, after mixing, take 0.975g of crude enzyme solution (enzyme concentration 5U/mL), 0.24g of sodium sulfate, after mixing, add 0.285g of poly Ethylene glycol 400 and 0.3g of n-hexane solution containing 0.5% 6-methyl 5-heptenyl 2-alcohol acetate, filled into a stoppered flask and mixed evenly, and placed on a constant temperature shaker with a rotating speed of 9000rpm. Control at 45 °C for 20min.
  • CALB lipase Take an appropriate amount of CALB lipase in an Erlenmeyer flask, add 100 times the volume of water, after mixing, take 0.975g of crude enzyme solution (enzyme concentration 5U/mL), 0.24g of sodium sulfate, after mixing, add 0.285g of poly Ethylene glycol 400 and 0.3g of n-hexane solution containing 0.5% 1-(4-methylphenyl) ethanol acetate, filled in a stoppered flask, mixed evenly, and placed on a constant temperature shaker with a rotation speed of 9000rpm, controlled React for 20 min at 45°C.
  • optical rotation and enantioselectivity E of 1-(4-methylphenyl)ethanol in the available system reached 95.87% and 156.87, respectively, while the control group was only divided into upper and lower phases, and both phases of the product were distributed.
  • the optical rotation and enantioselectivity E of the product are only 95.45% and 76.18.
  • lipase AY30 in an Erlenmeyer flask, add 100 times the volume of water, mix it, take 0.9g of crude enzyme solution (enzyme concentration 100U/mL), 0.3g of ammonium sulfate, add 0.3g of polymer after mixing Ethylene glycol 400 and 0.3g of isooctane solution containing 0.5% mixed naproxen methyl ester, filled in a stoppered flask and mixed evenly, and placed on a constant temperature shaker with a rotation speed of 200rpm, controlled at 37°C Reaction 4h.
  • the optical rotation and enantioselectivity E values of R-naproxide production in the system reached 89.9% and 28, respectively, while the control group was only divided into upper and lower phases, and the product was distributed in both phases.
  • the E values of enantioselectivity are only 86.5% and 18.
  • lipase AY30 Take an appropriate amount of lipase AY30 in an Erlenmeyer flask, add 20 volumes of phosphate buffer solution (100 mM) with a pH value of 8, after mixing, take 0.96g of crude enzyme solution (enzyme concentration 500U/mL), 0.18g Sodium sulfate, after mixing, add 0.36g of polyethylene glycol 400 and 0.3g of isooctane solution containing 0.5% of mixed naproxen methyl ester, put it into a triangular flask with a stopper, mix well, and put it at the speed of A constant temperature shaker at 200 rpm was used to control the reaction at 37°C for 4 hours.
  • Lipase is mainly distributed in the middle liquid layer.
  • the pH value is 8
  • the distribution coefficient of the middle and lower liquid layer can reach 88
  • the control group is only 62 and 42
  • the optical rotation of the R-naprox production in the system can reach 98.5%.
  • the conversion rate can reach 32%, and when the pH value is 5, the optical rotation is only 92%.
  • the pH value is 10 although a similar optical rotation can be obtained, the conversion rate is only 12%.

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Abstract

生物工程与食品技术领域，涉及一种酶法拆分手性物质的方法，包括以下步骤：(1)配置脂肪酶浓度为1-3000U/mL的酶液，向酶液中添加可溶性盐、亲水性溶剂和疏水性溶剂，配成三液相体系；所述疏水性溶剂中含有消旋型手性物构成的酯类或酰胺类化合物；(2)将三液相体系于搅拌条件下进行酶催化反应，反应结束后，静置或离心至分为三层，从上自下依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层或下液层，上液层产物为另一含单一旋光手性产物的酯类或酰胺类产品。该方法具有能耗小、原料利用率高、反应条件温和等优点，解决了现有酶法手性拆分效率低、手性选择性差、回收率低、难于工业化的难题。

Description

一种酶法拆分手性物质的方法 技术领域
本发明属于生物工程与食品技术领域，涉及到酶的分离与应用技术，特别涉及到利用酶法拆分手性物质的方法。
 背景技术
近几十年，手性物质开发处于井喷发展期，世界上正在开发的药物中，三分之二以上是手性药物，目前其市场规模已超过2500亿美金。而以药效更高、副作用更小的单一手性药物取代传统混合消旋体药物已成为发展的必然趋势。我国虽然在手性物质上拥有大量的需求缺口和相关的基础资源，但由于技术落后,产品质量差等问题导致绝大部分相关产品难于与西方国家竞争，亟需产业升级。
目前单一构型异构体的制备主要通过诱导结晶或化学拆分法等方法对外消旋体的拆分，传统工艺通常采用手性拆分剂拆分，效率低，要经过多次反复精制才能达到标准。导致整个过程工艺极端复杂，成本极高等难题（J. Mol. Catal. B: Enzym., 2010, 62(2): 162−168, Org. Biomol. Chem., 2014, 12, 6634-6642, J. Mol. Catal. B: Enzym., 2010, 65(1/4):49−51）。而酶法拆分具有条件温和、产品纯度高等优点，被寄予厚望，但是，一方面，手性产物与其对映体副产物的理化性质过于接近，使其难于通过常规分离手段将产物与副产物同步分离，来提高反应选择性。通过加入手性选择剂的萃取拆分虽有一定潜力，但脂肪酶催化属于界面催化，即使在双相体系中，其产物也会对反应具有较强的抑制作用，因而，难于利用现有体系解除产物抑制以提高催化效率。另一方面，手性拆分对产物的光学纯度要求又极高，通常情况下，单旋光性产物的纯度通常要大于99%，随着反应进行，目标底物含量逐渐降低，产物逐渐积累，导致大部分反应副反应多、产物纯度低、转化率差等难题，因此仅在少数反应具有高选择性的酶（通常E≥100）才有工业化潜力，这就极大地加大了其研发难度(Tetrahedron 2007, 63: 1721–1754)。另外，传统酶催化体系还存在控制困难，产物分离及酶的回用往往难于兼容等难题，严重制约了其发展。因此亟需开发高效、可控的催化及分离体系，以适用其发展（Journal of Molecular Catalysis B: Enzymatic, 2012, 4:78-82，Tetrahedron 2007, 63: 1721–1754) 。
 技术问题
一种酶法拆分手性物质的方法，解决了现有酶法手性拆分效率低、成本高、手性选择性差、回收率低、难于工业化的难题。
 技术解决方案
本发明的目的是针对目前酶法拆分生产手性物质过程中，存在的成本高，连续化生产困难，产物抑制带来的反应效率低、反应时间长等问题，提供了一种高效快速的酶法拆分生产手性物质的方法。
本发明的目的通过以下技术方案实现：
一种酶法拆分手性物质的方法，包括以下步骤：
（1）配置脂肪酶浓度为1-3000U/mL 的酶液，向酶液中添加可溶性盐、亲水性溶剂和疏水性溶剂，配成三液相体系；所述可溶性盐、亲水性溶剂和疏水性溶剂与酶液的质量比分别为0.1-0.9、0.1-5和0.1-10；所述疏水性溶剂中含有消旋型手性物构成的酯类或酰胺类化合物；
（2）将三液相体系于搅拌条件下进行酶催化反应，反应结束后，静置或离心至分为三层，从上自下依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层或下液层，上液层产物为另一含单一旋光手性产物的酯类或酰胺类产品。
优选地，步骤（1）所述亲水性溶剂为聚乙二醇、聚丙二醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、乙二醇和丙酮中的一种或两种以上；或者所述亲水性溶剂为[BMIM]Br、[BMIM]BF 4、[EMIM]ETSO 4和[OMIM]Cl中的一种或两种以上。
优选地，步骤（1）所述可溶性盐是柠檬酸钠、氯化钠、硫酸铵、碳酸钠、磷酸氢二钾、磷酸钾、磷酸二氢钾和磷酸氢二钾中的一种或两种以上。
优选地，步骤（1）所述疏水性溶剂是正己烷、乙醚、异丙醚、乙酸乙酯、环己醇、石油醚、异辛烷、苯和甲苯中的一种或两种以上。
优选地，步骤（2）的反应条件为：温度30~45℃，反应时间为20min~4h。
反应操作一般在低于溶剂易挥发的温度下进行。为了使手性产物的脱除和改性效果尽可能达到最佳，可以调节体系的pH值。优选地，步骤（1）所述三液相体系的pH值为5-9。
优选地，步骤（1）所述脂肪酶浓度为5-2000U/mL。
优选地，所述可溶性盐、亲水性溶剂和疏水性溶剂与酶液质量比分别为0.2-0.8、0.2-0.8和0.2-4。
优选地，所述消旋型手性物构成的酯类或酰胺类化合物选自混旋型扁桃酸甲酯、混旋型萘普生甲酯、混旋型（4-甲氧基-苯基）-1-乙醇乙酸酯、混旋型1-(4-甲氧基苯基)乙醇乙酸酯、混旋型6-甲基5-庚烯基2-醇乙酸酯中的任意一种或两种以上。
优选地，所述消旋型手性物构成的酯类或酰胺类化合物占疏水性溶剂质量的0.1-10%，，优选1%~5%。
上述脂肪酶催化反应中，脂肪酶可以来自天然，也可以通过人工发酵产生。可以是发酵液，可以是经简单纯化后的粗酶，也可是经纯化后纯酶。
 有益效果
本发明的有益效果如下：
本发明克服了目前酶法拆分并同步回收手性物质过程中，存在的成本高，连续化生产困难，产物抑制带来的反应效率低、反应时间长等难题，提供了一种从粗酶液中分离脂肪酶以及利用脂肪酶拆分生产手性物质的方法。使工艺得以简化，分离时间短，成本降低，经济可行。具体表现在：利用三液相体系可将一种单一旋光手性产物的酯类或酰胺类底物分配在疏水相，脂肪酶分配在富溶剂相或富盐相，水解后的单一旋光手性产物主要富集于另一相，经简单离心或静置等方式即可将产物回收利用。该过程既有利于酶的纯化与回收，也有提高催化效率，大幅降低酶的损耗与纯化成本。该方法具有能耗小、原料利用率高、反应条件温和等优点，解决了现有酶法拆分生产手性物质难于工业化的技术难题。
 本发明的实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
本实施例中本实施例中所用CALB（Candida antarctica lipase B），Novozyme-51032（cutinase）脂肪酶购于Novozyme公司，脂肪酶AY30（Candida rugosa lipases）购于日本天野公司。
实施例1
取适量CALB脂肪酶置于锥形瓶中，加入1000倍体积的水，混匀后，取1g粗酶液（酶浓度5U/mL），0.6g 的磷酸氢二钾，混匀后加入0.4g聚乙二醇400和0.4g含5%混旋型扁桃酸甲酯的异丙醚溶液，装入具塞三角瓶中混合均匀，混合溶液的pH值为8.9，并置于转速为200rpm的恒温摇床上，控制在30℃下反应2h。另取1g粗酶液（5U/mL），加入0.4g含5%混旋型扁桃酸甲酯的异丙醚溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定5000rpm转速离心5min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层或下液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。脂肪酶主要分配于中液层，中下液层的分配系数可达4.75。另取中下液层测量其酶活和上液层扁桃酸甲酯的旋光度，可得疏水相中S-扁桃酸甲酯的旋光度达到了99.5%，而对照组仅分为上下两相，产物两相均有分布，S-扁桃酸甲酯总的旋光度仅为43%。
实施例 2
取适量脂肪酶AY30置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度100U/mL），0.225g 的硫酸钠，混匀后加入0.3g聚乙二醇400和0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，装入具塞三角瓶中混合均匀，混合溶液的pH值为7.0，并置于转速为200rpm的恒温摇床上，控制在37℃下反应4h。另取0.975g粗酶液（200U/mL），加入0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定3000rpm转速离心1min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。脂肪酶主要分配于中液层，中下液层的分配系数可达48.9。体系中R-萘普生产物的旋光度及对映体选择率E值分别达到了92.9%和46，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为86.5%和18。
实施例3
取适量脂肪酶AY30置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度200U/mL），0.24g 的硫酸钠，混匀后加入0.285g聚乙二醇600和0.3g含0.5%混旋型（4-甲氧基-苯基）-1-乙醇乙酸酯的异辛烷溶液，装入具塞三角瓶中混合均匀，混合溶液的pH值为7.0，并置于转速为200rpm的恒温摇床上，控制在45℃下反应20min。另取0.975g粗酶液（100U/mL），加入0.3g含0.5%混旋型（4-甲氧基-苯基）-1-乙醇乙酸酯的异辛烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定9000rpm转速离心3min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。脂肪酶主要分配于中液层，中下液层的分配系数可达149.1，体系中（4-甲氧基-苯基）-1-乙醇的旋光度和对映体选择率E值分别达到了98.13%和232.8，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为94.13%和69.39。
实施例 4
取适量脂肪酶AY30置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度100U/mL），0.225g 的硫酸钠，混匀后加入0.3g [BMIm]BF 4和0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在37℃下反应4h。反应结束后，设定3000rpm转速离心1min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类产品。脂肪酶主要分配于下液层，下液层的酶回收率可达98.5%。体系中R-萘普生产物的旋光度及对映体选择率E值亦分别达到了82.1%和11。
实施例5
取适量Novozyme-51032脂肪酶置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度5U/mL），0.24g 的硫酸钠，混匀后加入0.285g聚乙二醇400和0.3g含0.5%混旋型1-(4-甲氧基苯基)乙醇乙酸酯的正己烷溶液，装入具塞三角瓶中混合均匀，混合溶液的pH值为7.0，并置于转速为9000rpm的恒温摇床上，控制在45℃下反应20min。另取0.975g粗酶液（5U/mL），加入0.3g含0.5%混旋型1-(4-甲氧基苯基)乙醇乙酸酯正己烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定9000rpm转速离心3min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。另取中下液层测量其酶活和上液层1-(4-甲氧基苯基)乙醇的旋光度，可得疏水相中1-(4-甲氧基苯基)乙醇的旋光度和对映体选择率E分别达到了96.07%和80.96，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为85.72%和 17.01。
实施例6
取适量CALB脂肪酶置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度5U/mL），0.24g 的硫酸钠，混匀后加入0.285g聚乙二醇400和0.3g含0.5% 6-甲基5-庚烯基2-醇乙酸酯正己烷溶液，装入具塞三角瓶中混合均匀，并置于转速为9000rpm的恒温摇床上，控制在45℃下反应20min。另取0.975g粗酶液（5U/mL），加入0.3g含0.5%混旋型6-甲基5-庚烯基2-醇乙酸酯的正己烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定9000rpm转速离心3min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。另取中下液层测量其酶活和上液层6-甲基5-庚烯基2-醇的旋光度，可得疏水相中6-甲基5-庚烯基2-醇的旋光度和对映体选择率E值分别达到了96.49%和72.85，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为88.09%和18.47。
实施例7
取适量CALB脂肪酶置于锥形瓶中，加入100倍体积的水，混匀后，取0.975g粗酶液（酶浓度5U/mL），0.24g 的硫酸钠，混匀后加入0.285g聚乙二醇400和0.3g含0.5% 1-(4-甲基苯基)乙醇乙酸酯正己烷溶液，装入具塞三角瓶中混合均匀，并置于转速为9000rpm的恒温摇床上，控制在45℃下反应20min。另取0.975g粗酶液（5U/mL），加入0.3g含0.5%混旋型1-(4-甲基苯基)乙醇乙酸酯的正己烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定9000rpm转速离心3min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。可得体系中1-(4-甲基苯基)乙醇的旋光度和对映体选择率E值分别达到了95.87%和156.87，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为95.45%和76.18。
实施例 8
取适量脂肪酶AY30置于锥形瓶中，加入100倍体积的水，混匀后，取0.9g粗酶液（酶浓度100U/mL），0.3g 的硫酸铵，混匀后加入0.3g聚乙二醇400和0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在37℃下反应4h。另取0.975g 粗酶液（100U/mL），加入0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，并用水稀释至相同体积，在相同条件下反应作为对照。反应结束后，设定3000rpm转速离心1min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。脂肪酶主要分配于中液层，中下液层的分配系数可达21.4。体系中R-萘普生产物的旋光度及对映体选择率E值分别达到了89.9%和28，而对照组仅分为上下两相，产物两相均有分布，产物的旋光度和对映体选择率E值仅为86.5%和18。
实施例 9
取适量脂肪酶AY30置于锥形瓶中，加入20倍体积的pH值为8的磷酸盐缓冲液（100mM），混匀后，取0.96g粗酶液（酶浓度500U/mL），0.18g 的硫酸钠，混匀后加入0.36g聚乙二醇400和0.3g含0.5%混旋型萘普生甲酯的异辛烷溶液，装入具塞三角瓶中混合均匀，并置于转速为200rpm的恒温摇床上，控制在37℃下反应4h。另取取适量脂肪酶AY30置于锥形瓶中，分别加入20倍体积的pH值为5和10的磷酸盐缓冲液（100mM），分别取0.96g粗酶液（500U/mL），按上述方法配成相同比例但pH值不同的三液相体系，在相同条件下反应作为对照。反应结束后，设定3000rpm转速离心1min，分为三层，依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层，收集上液层产物即为另一单一旋光手性产物的酯类或酰胺类产品。脂肪酶主要分配于中液层，pH值为8时，中下液层的分配系数可达88，而对照组仅为62和42，体系中R-萘普生产物的旋光度可达98.5%，转化率可达32%，而pH值为5时，其旋光度仅为92%，而当pH值为10时，虽可获得相似的旋光度，但其转化率仅为12%。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种酶法拆分手性物质的方法，其特征在于，包括以下步骤：
    （1）配置脂肪酶浓度为1-3000U/mL 的酶液，向酶液中添加可溶性盐、亲水性溶剂和疏水性溶剂，配成三液相体系；所述可溶性盐、亲水性溶剂和疏水性溶剂与酶液的质量比分别为0.1-0.9、0.1-5和0.1-10；所述疏水性溶剂中含有消旋型手性物构成的酯类或酰胺类化合物；
    （2）将三液相体系于搅拌条件下进行酶催化反应，反应结束后，静置或离心至分为三层，从上自下依次为上液层、中液层和下液层，水解后的单一旋光手性产物主要富集于中液层或下液层，上液层产物为另一含单一旋光手性产物的酯类或酰胺类产品。
  2. 根据权利要求1所述的方法，其特征在于，步骤（1）所述亲水性溶剂为聚乙二醇、聚丙二醇、乙醇、正丙醇、异丙醇、正丁醇、异丁醇、乙二醇和丙酮中的一种或两种以上；或者所述亲水性溶剂为[BMIM]Br、[BMIM]BF 4、[EMIM]ETSO 4和[OMIM]Cl中的一种或两种以上。
  3. 根据权利要求1所述的方法，其特征在于，步骤（1）所述可溶性盐是柠檬酸钠、氯化钠、硫酸铵、碳酸钠、磷酸氢二钾、磷酸钾、磷酸二氢钾和磷酸氢二钾中的一种或两种以上。
  4. 根据权利要求1所述的方法，其特征在于，步骤（1）所述疏水性溶剂是正己烷、乙醚、异丙醚、乙酸乙酯、环己醇、石油醚、异辛烷、苯和甲苯中的一种或两种以上。
  5. 根据权利要求1或2或3或4所述的方法，其特征在于，步骤（2）的反应条件为：温度30~45℃，反应时间为20min~4h。
  6. 根据权利要求5所述的方法，其特征在于，步骤（1）所述三液相体系的pH值为5-9。
  7. 根据权利要求6所述的方法，其特征在于，步骤（1）所述脂肪酶浓度为5-2000U/mL。
  8. 根据权利要求7所述的方法，其特征在于，所述可溶性盐、亲水性溶剂和疏水性溶剂与酶液质量比分别为0.2-0.8、0.2-0.8和0.2-4。
  9. 根据权利要求1或2或3或4所述的方法，其特征在于，所述消旋型手性物构成的酯类或酰胺类化合物选自混旋型扁桃酸甲酯、混旋型萘普生甲酯、混旋型（4-甲氧基-苯基）-1-乙醇乙酸酯、混旋型1-(4-甲氧基苯基)乙醇乙酸酯、混旋型6-甲基5-庚烯基2-醇乙酸酯中的任意一种或两种以上。
  10. 根据权利要求1或2或3或4所述的方法，其特征在于，所述消旋型手性物构成的酯类或酰胺类化合物占疏水性溶剂质量的0.1%-10%，优选1%~5%。
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