阅读说明：本技术 一种脂肪酶固定化新技术及其应用于对映体拆分的方法 (New lipase immobilization technology and method for applying same to enantiomer resolution ) 是由 唐课文 欧剑 袁欣 许卫凤 戴桂林 张盼良 于 2020-06-30 设计创作，主要内容包括：本专利介绍了一种采用物理吸附法制备固定化酶(PCL@ZIF-8),并将其应用于催化水解2-苯基丙酸异丁酯制备(S)-2-苯基丙酸和酯交换外消旋1-苯乙醇制备(S)-1-苯乙醇。在选择性催化水解2-苯基丙酸异丁酯中,反应前2 h,固定化酶的反应速率为游离脂肪酶的3倍,反应平衡时,底物的单一转化率为92.33%,产物光学纯度为99.62%,循环4次后,固定化后的脂肪酶仍保持了良好的催化活性。在选择性催化酯交换1-苯乙醇中,固定化脂肪酶催化效率比游离酶略高,且展现了优异的重复使用性能,循环6次后,依然保持其初始活性的69.33%。与游离脂肪酶相比,该固定化酶展现了优异的催化效率,且具有良好的重复使用性能。MOFs具有合成简单、种类丰富、稳定性好等优点,是一种潜在的固定化酶载体,在工业应用中具有广阔的前景。(The patent introduces immobilized enzyme (PCL @ ZIF-8) prepared by adopting a physical adsorption method, and the immobilized enzyme is applied to the preparation of (S) -2-phenylpropionic acid by catalyzing and hydrolyzing isobutyl 2-phenylpropionate and the preparation of (S) -1-phenethyl alcohol by ester exchange racemization 1-phenethyl alcohol. In the selective catalytic hydrolysis of 2-isobutyl phenylpropionate, the reaction rate of immobilized enzyme is 3 times of that of free lipase 2 hours before reaction, when the reaction is balanced, the single conversion rate of a substrate is 92.33%, the optical purity of a product is 99.62%, and the immobilized lipase still maintains good catalytic activity after 4 times of circulation. In the selective catalytic transesterification 1-phenylethyl alcohol, the catalytic efficiency of the immobilized lipase is slightly higher than that of the free enzyme, the immobilized lipase shows excellent reusability, and the immobilized lipase still keeps 69.33 percent of the initial activity after being cycled for 6 times. Compared with free lipase, the immobilized enzyme shows excellent catalytic efficiency and has good reusability. MOFs have the advantages of simple synthesis, rich varieties, good stability and the like, are potential immobilized enzyme carriers, and have wide prospects in industrial application.)

1. A method for immobilizing lipase applied to chiral enantiomer resolution is characterized in that the lipase is immobilized in a cavity of a zeolite imidazole framework material (ZIF-8) by a physical adsorption method, and the immobilized enzyme is used for catalyzing hydrolysis resolution of a 2-phenylpropionic acid enantiomer and ester exchange resolution of a 1-phenethyl alcohol enantiomer.

2. The method according to claim 1, characterized in that it comprises the following operating steps:

in the process of synthesizing the immobilized enzyme, selecting a proper concentration of an enzyme solution, adding a certain amount of ZIF-8, oscillating and reacting for a certain time at a certain temperature, filtering and freeze-drying after the reaction is finished to obtain the immobilized enzyme; in the catalytic hydrolysis and resolution of 2-phenylpropionic acid enantiomer by immobilized enzyme, selecting phosphate buffer with proper pH value as reaction medium, adding a certain amount of racemic 2-phenylpropionic acid isobutyl ester, adding a certain amount of immobilized enzyme as catalyst into a reaction tube, stirring and reacting for a certain time at a certain temperature, and detecting a certain volume of reaction liquid after the reaction is finished; an organic solvent is selected as a reaction medium in the 1-phenethyl alcohol enantiomer separated by the immobilized enzyme catalysis ester exchange, a certain amount of racemic 1-phenethyl alcohol and vinyl acetate are added, a certain amount of immobilized enzyme is added into a reaction tube as a biocatalyst, the mixture is stirred and reacted for a certain time at a certain temperature, after the reaction is finished, a certain volume of reaction liquid is diluted, and a sample is taken for detection.

3. The method of claim 2, wherein the concentration of the enzyme solution is 2-20 mg/mL during the synthesis of the immobilized enzyme.

4. The method of claim 2, wherein the concentration of ZIF-8 ranges from 1 to 10 mg/mL during the synthesis of the immobilized enzyme.

5. The method of claim 2, wherein the immobilization reaction temperature is 5-40 ℃ during the synthesis of the immobilized enzyme^oC。

6. The method of claim 2, wherein the adsorption oscillation time is 12-24 h during the synthesis of the immobilized enzyme.

7. The process of claim 2, wherein in the resolution of 2-phenylpropionic acid enantiomer, the concentration of 2-phenylpropionic acid isobutyl ester is 5-20 mmol/L, the concentration of immobilized enzyme is 2-20 mg/mL, the pH range is 3-8, and the reaction temperature is 25-70^oAnd C, the reaction time is 1-40 h.

8. The method as claimed in claim 2, wherein in the resolution of 1-phenylethyl alcohol enantiomer, the concentration of 1-phenylethyl alcohol is 10-40 mmol/L, the concentration of vinyl acetate is 100-600mmol/L, the concentration of immobilized enzyme is 2-20 mg/mL, the reaction temperature is 20-50^oAnd C, the reaction time is 1-30 h.

9. The method of claim 2, wherein the immobilized enzyme is reusable and the steps of: after the reaction is finished, the immobilized enzyme is separated from the reaction medium by filtration, and the immobilized enzyme is reused after freeze drying.

Technical Field

The invention belongs to the technical field of biocatalysis, and provides an optical homochiral compound prepared by a biocatalysis method, wherein a physical adsorption method is used for fixing free Pseudomonas Cepacia Lipase (PCL) on a zeolite imidazole framework material (ZIF-8) to prepare an immobilized enzyme (PCL @ ZIF-8). PCL @ ZIF-8 is used as a biocatalyst to carry out stereoselective catalytic hydrolysis resolution on the 2-phenylpropionic acid enantiomer and transesterification resolution on the 1-phenethyl alcohol enantiomer.

Background

The 2-aryl propionic acid medicine is one kind of non-steroidal anti-inflammatory medicine and has excellent anti-inflammatory, antipyretic and analgesic effects. Generally, the (S) -enantiomer has higher pharmacological activity and fewer side effects than the (R) -enantiomer. For example, (S) -ibuprofen has 100 times of the anti-inflammatory and analgesic effects of (R) -ibuprofen; the drug effect of (S) -flurbiprofen is 30 times that of (R) -flurbiprofen. 2-phenylpropionic acid (PPA) is an important intermediate for synthesizing various 2-aryl propionic acid medicaments, so that chiral resolution of PPA enantiomer has important significance for preparing optically pure (S) -2-aryl propionic acid medicaments. The chiral alcohol is a kind of medicine for synthesizing several diseases of angiocardiopathy and hypertension, etc. 1-phenylethyl alcohol is an important precursor for drug synthesis. (S) -1-phenylethyl alcohol can be used for synthesizing sertraline hydrochloride, treating depression related symptoms, and can be used for synthesizing asthma and immunity enhancing medicine. The (R) -1-phenethyl alcohol can be used for synthesizing medicaments for inhibiting the absorption of cholesterol. Thus, the preparation of a single 1-phenylethyl alcohol enantiomer is of great significance in the pharmaceutical industry.

The preparation method of the single enantiomer mainly comprises an asymmetric synthesis method and a racemate resolution method. The asymmetric synthesis method is to synthesize a target single enantiomer by controlling reaction conditions. The racemate resolution method is to obtain a single enantiomer by separating the racemate, and comprises a crystallization resolution method, a chromatography method, a membrane resolution method, a chiral solvent extraction method and the like. Asymmetric synthesis methods have the advantages that chiral pure enantiomers can be directly obtained, atom economy is high, but generally, asymmetric synthesis methods need chiral sources, chiral auxiliaries and chiral reagents, economic cost is high, environmental dependence is high, and optical purity of synthesized products is not high. Compared with an asymmetric synthesis method, the racemate resolution method mainly realizes the high-efficiency resolution of the racemate by methods such as physics, chemistry or biology, and the like, so that a single enantiomer with high optical purity is obtained, the development time is short, the operation is simple and convenient, the operation cost is low, and the resolution method is mainly adopted for industrially preparing the single enantiomer at present. The enzyme resolution method has the advantages of mild conditions, high selectivity, less side reactions, less impurity components, high yield, simple reaction operation and the like, and is less in pollution, so that the influence of chemical production on the environment is reduced to a great extent, and the method conforms to the green chemical concept. At present, the most common method for industrially preparing optically pure alcohol and amine compounds is an enzyme resolution method, but free enzyme has the defects of difficult separation, poor stability, incapability of being repeatedly used and the like. The enzyme immobilization technology can effectively improve the stability of the enzyme, enhance the activity of the enzyme and expand the operation range, and is one of effective strategies for solving the defect of free enzyme.

Immobilization may result in changes in the physicochemical properties of the enzyme, and the carrier and method of immobilization chosen are key factors in causing these changes. Immobilization of enzymes has been widely reported, and many carriers are available, such as sol-gel matrix, hydrogel, organic microparticles, mesoporous silica, and the like. However, these carriers have certain defects, such as easy enzyme denaturation caused by sol-gel matrix and limited mass transfer of the substrate in the sol-gel matrix; because the hydrogel and the organic particles are easy to expand and degrade, the enzyme fixed on the hydrogel and the organic particles is easy to lose and denature, and the mass transfer efficiency is low; although the mesoporous silica has various advantages, the mesoporous silica also has the problems that the structure cannot be reasonably designed, the surface is easy to change, and thus enzyme is denatured or lost, and the like.

Compared with the above-mentioned carriers, metal organic framework compounds (MOFs) are increasingly gaining attention as a new type of immobilized carriers. The MOFs have the advantages of high specific surface area, pore volume, pore channel size easy to adjust, metal nodes and ligands convenient to adjust, good thermal stability, milder synthesis conditions and the like. Meanwhile, as the nodes and the ligands can form abundant and diverse geometric connections, the MOFs have abundant and diverse structures, and the structures can be designed and adjusted according to specific applications. ZIF-8 is one of zeolitic imidazole framework materials, having a zeolitic topology. Compared with the common MOFs, ZIF-8 has more excellent thermal stability and aqueous solution stability besides the basic advantages of the MOFs.

The invention utilizes a physical adsorption method to fix Pseudomonas cepacia lipase in a ZIF-8 cavity (PCL @ ZIF-8), utilizes the excellent catalytic activity and stereoselectivity of the PCL @ ZIF-8 to catalyze, hydrolyze and split raceme 2-isobutyl phenylpropionate in a water phase, and prepares (S) -2-phenylpropionic acid and (R) -2-isobutyl phenylpropionate (formula 1) with high optical activity; in addition, (S) -1-phenylethyl alcohol and (R) -1-phenylethyl acetate (formula 2) having high optical activity were prepared by cleaving racemic 1-phenylethyl alcohol by catalytic transesterification using PCL @ ZIF-8 in an organic phase. According to the method, activity of PCL @ ZIF-8 is compared with that of free PCL, and the PCL @ ZIF-8 has higher catalytic activity and keeps high stereoselectivity. The PCL @ ZIF-8 has excellent dispersibility and stability in both aqueous and organic phases, is easy to separate and reuse, and remarkably reduces the production cost. The technology solves the problems that the free enzyme can not be reused in an enzyme resolution method, the product and the enzyme are difficult to separate, and the reaction time is long;

formula 1. lipase catalyzed resolution of isobutyl 2-phenylpropionate enantiomer

And (2) carrying out lipase catalytic resolution on the 1-phenethyl alcohol enantiomer.

Disclosure of Invention

The invention provides a method for immobilizing Pseudomonas cepacia lipase in a ZIF-8 cavity (PCL @ ZIF-8) by adopting a physical adsorption method, and applying the PCL @ ZIF-8 to catalytic hydrolysis resolution of a 2-phenylpropionic acid enantiomer and ester exchange resolution of a 1-phenethyl alcohol enantiomer. The catalytic performance was studied in both systems, obtaining excellent yields and purities. After the free lipase is immobilized, the thermal stability, catalytic activity and reusability of the lipase are obviously improved.

The technical scheme of the invention is as follows: zn (NO) is used as the base material in the invention₃)₂•6H₂O is a metal ion center, 2-methylimidazole is an organic ligand, and DMF is a reaction medium, so that a ZIF-8 crystal is prepared; and then immobilizing the Pseudomonas cepacia lipase in a ZIF-8 cavity by a physical adsorption method in a phosphate buffer by taking ZIF-8 as an immobilized carrier. In the reaction for hydrolyzing and resolving 2-phenylpropionic acid enantiomer, phosphate buffer is used as a reaction medium, racemic isobutyl 2-phenylpropionate is used as a substrate, the concentration of the isobutyl 2-phenylpropionate enantiomer is 5-20 mmol/L, 2-20 mg/mL PCL @ ZIF-8 is added as a biocatalyst, and the temperature is 25-70 DEG C^oAnd C, stirring and heating for reaction for a certain time. After the reaction is finished, a certain amount of samples are taken to carry out qualitative and quantitative detection on the product through a high performance liquid chromatograph, and the substrate conversion rate and the enantiomeric excess are calculated. In the ester exchange resolution 1-phenethyl alcohol enantiomer reaction, n-hexane is taken as a reaction medium, racemic 1-phenethyl alcohol and vinyl acetate are taken as reaction substrates, the substrates are dissolved in the n-hexane, the concentration of the 1-phenethyl alcohol is 10-40 mmol/L, the concentration of the vinyl acetate is 100-600mmol/L, 2-20 mg/mL PCL @ ZIF-8 is added as a biocatalyst, and the temperature range is 20-50^oAnd C, stirring and heating the mixture in a closed reaction tube system of 25mL for reaction for a certain time. After the reaction is finished, a certain amount of samples are taken to carry out qualitative and quantitative detection on the product through a high performance liquid chromatograph, and the substrate conversion rate and the enantiomeric excess are calculated.

Compared with the prior art, the invention has the following advantages:

the invention successfully prepares the PCL @ ZIF-8 by using mesoporous ZIF-8 as an immobilized carrier and adopting a simple physical adsorption method. Because the PCL @ ZIF-8 is a porous material, the PCL @ ZIF-8 can adsorb a substrate and reduce mass transfer resistance, when the PCL @ ZIF-8 is used as a reaction catalyst, the defects of instability, low activity, difficulty in separation from a product and the like of lipase can be overcome, and a target product with high yield and high purity can be obtained. Meanwhile, the PCL @ ZIF-8 is insoluble in both the water phase and the organic phase, so that the PCL @ ZIF-8 has good reusability. The method is simple and convenient to implement and operate, green and environment-friendly, products are easy to separate, and the reuse rate of the catalyst is high.

[ detailed description ] according to the present embodiment

The method comprises the following specific steps:

first, testing and analyzing

In the reaction of 2-phenylpropionic acid enantiomer resolution by hydrolysis, the optical purity and the substrate conversion rate of the product are analyzed by a United states Waters 1525 high performance liquid chromatograph, Inertsil ODS-3 column (250 mm × 4.6.6 mm, 5 μm) and mobile phase composition is V (acetonitrile), V (water) = 20: 80 flow rate is 1 mL/min, UV detection wavelength is 225 nm, and column temperature is 25.0^oC, sample size of 10 μ L. In the reaction of ester exchange resolution of 1-phenethyl alcohol enantiomer, the optical purity and the substrate conversion rate of the product are analyzed by a United states Waters 1525 high performance liquid chromatograph, Chiralcel^®OJ-RH chiral column (250 mm × 4.6.6 mm, 5 μm). The mobile phase composition is V (methanol) = V (water) = 20: 80, flow rate is 0.5 mL/min, UV detection wavelength is 210nm, column temperature is 25.0^oC, sample size of 10 μ L.