阅读说明：本技术 纳米高分子-双酶复合物及其制备与在（r）-2-羟基-4-苯基丁酸乙酯合成中的应用 (Nano polymer-double enzyme compound, preparation thereof and application thereof in synthesis of (R) -2-hydroxy-4-phenyl ethyl butyrate ) 是由 欧志敏 卢媛 程朋朋 代洪倩 王金美 张楚玥 唐岚 于 2020-11-24 设计创作，主要内容包括：本发明公开了一种纳米高分子-双酶复合物及其制备与在(R)-2-羟基-4-苯基丁酸乙酯合成中的应用,以葡萄糖脱氢酶和来源于Saccharomyces cerevisiae CGMCC No.3361的羰基还原酶为双酶,以聚丙烯酸为载体,以1-乙基-3-(3-二甲基氨基丙基)碳二亚胺为交联剂,以磷酸盐缓冲液为反应介质,在20℃-40℃、180-220rpm摇床中混合搅拌10-60min,获得纳米高分子-双酶复合物。本发明高分子-双酶复合物用于油水两相中羰基化合物的不对称还原反应,可以同时实现辅酶再生,该方法可以提高催化剂的稳定性,延长催化剂使用寿命,实现重复利用,提高生产效率。(The invention discloses a nanometer polymer-double enzyme compound and a preparation method thereof and application thereof in (R) -2-hydroxy-4-phenyl ethyl butyrate synthesis, wherein glucose dehydrogenase and carbonyl reductase derived from Saccharomyces cerevisiae CGMCC No.3361 are used as double enzymes, polyacrylic acid is used as a carrier, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is used as a cross-linking agent, phosphate buffer is used as a reaction medium, and the mixture is mixed and stirred for 10-60min in a shaking table with the temperature of 20-40 ℃ and the rotational speed of 180-plus 220rpm to obtain the nanometer polymer-double enzyme compound. The macromolecule-double enzyme compound is used for asymmetric reduction reaction of carbonyl compounds in oil-water two phases, can realize coenzyme regeneration at the same time, and can improve the stability of the catalyst, prolong the service life of the catalyst, realize reutilization and improve the production efficiency.)

1. A nanometer macromolecule-double enzyme compound is characterized in that the nanometer macromolecule-double enzyme compound is prepared by the following method: mixing and stirring carbonyl reductase and glucose dehydrogenase as double enzymes, polyacrylic acid as a carrier, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide as a cross-linking agent and phosphate buffer as a reaction medium in a shaking table at the temperature of 20-40 ℃ and the rotational speed of 180-220rpm for 10-60min to obtain a nano polymer-double enzyme compound; the carbonyl reductase is derived from Saccharomyces cerevisiae CGMCC No. 3361.

2. The biopolymer-bis-enzyme complex of claim 1, wherein the mass ratio of carbonyl reductase to glucose dehydrogenase is 1:0.5 to 1; the mass ratio of the total amount of the carbonyl reductase and the glucose dehydrogenase to the polyacrylic acid is 1: 1-5; the mass ratio of the total amount of the carbonyl reductase and the glucose dehydrogenase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20.

3. The biopolymer-bis-enzyme complex of claim 1, wherein the biopolymer-bis-enzyme complex is prepared by the following method: mixing and stirring polyacrylic acid solution, carbonyl reductase and glucose dehydrogenase for 10-60min in a shaking table with the rotation speed of 180-220rpm at the temperature of 20-40 ℃, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10mM phosphate buffer solution with the pH value of 7.4, mixing and stirring for 10-60min in a shaking table with the rotation speed of 180-220rpm at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with the pH value of 7.4, dialyzing by adopting a dialysis membrane with the pH value of 25kDa, and taking trapped fluid to obtain a nano polymer-double enzyme compound; the polyacrylic acid solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with equal volume of 10mM phosphate buffer solution with pH 7.4.

4. The biopolymer-bis-enzyme complex of claim 1, wherein the biopolymer-bis-enzyme complex is prepared by the following method: (1) mixing polyacrylic acid solution with 10mM phosphate buffer solution with pH 7.4, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring for 10-60min in a shaking table with the rotation speed of 180 plus of 220rpm at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with pH 7.4 of carbonyl reductase, mixing and stirring for 1-6h in a shaking table with the rotation speed of 180 plus of 220rpm at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with pH 7.4, dialyzing by adopting a dialysis membrane with 25kDa, and taking trapped fluid to obtain the carbonyl reductase-polyacrylic acid conjugate; the mass ratio of the carbonyl reductase to the polyacrylic acid is 1: 2-5; the mass ratio of the carbonyl reductase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20 parts of; the polyacrylic acid solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with equal volume of 10mM phosphate buffer solution with pH of 7.4; (2) mixing polyacrylic acid solution with 10mM phosphate buffer solution with pH 7.4, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring for 10-60min in a shaking table with the rotational speed of 180 plus at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with the pH 7.4, mixing and stirring for 1-6h in a shaking table with the rotational speed of 180 plus 220 at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with the pH 7.4, dialyzing by adopting a dialysis membrane with the molecular weight of 25kDa, and taking trapped fluid to obtain the glucose dehydrogenase-polyacrylic acid conjugate; the mass ratio of the glucose dehydrogenase to the polyacrylic acid is 1: 2-6; the mass ratio of the glucose dehydrogenase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20 parts of; the polyacrylic acid solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with equal volume of 10mM phosphate buffer solution with pH of 7.4; (3) mixing carbonyl reductase-polyacrylic acid conjugate, glucose dehydrogenase-polyacrylic acid conjugate and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring for 10-60min in a shaking table at the temperature of 20-40 ℃ and the rotational speed of 180-; the volume ratio of the carbonyl reductase-polyacrylic acid conjugate to the glucose dehydrogenase-polyacrylic acid conjugate is 1: 0.5-1.5; the mass ratio of the total mass of the carbonyl reductase-polyacrylic acid conjugate and the glucose dehydrogenase-polyacrylic acid conjugate to the mass of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1:0.5-1.

5. An application of the nano polymer-double enzyme compound of claim 1 in preparation of (R) -2-hydroxy-4-phenyl ethyl butyrate by asymmetric reduction of 2-oxo-4-phenyl ethyl butyrate.

6. The application of claim 5, wherein the application comprises the steps of taking 2-oxo-4-phenyl ethyl butyrate as a substrate, taking phosphate buffered saline solution with pH of 7.4 and 10mM as a reaction medium, taking d-glucose as an auxiliary substrate, taking NADP as a coenzyme, taking a nano polymer-double enzyme complex as a catalyst to form a conversion system, carrying out reduction reaction for 8-40 h at the temperature of 20-45 ℃ and at the rpm of 50-250, and separating and purifying a reaction solution to obtain the (R) -2-hydroxy-4-phenyl ethyl butyrate.

7. The use according to claim 6, wherein the substrate is added to a final concentration of 300 to 700 mmol/L; the final concentration of the d-glucose is 0.05-0.15M; the final concentration of the NADP is 0.03-0.07 mM; the final concentration of the catalyst is 0.1-0.6 mg/mL.

8. Use according to claim 6, characterized in that the reaction medium is a mixture of 10mM phosphate buffered saline solution, pH 7.4, and an organic solvent; the organic solvent is n-hexane, dibutyl phthalate, benzene, toluene or n-heptane.

9. Use according to claim 8, characterized in that the volume ratio of the 10mM phosphate buffered saline solution to the organic solvent, at pH 7.4, is between 1 and 5: 3.

10. use according to claim 8, characterized in that when the reaction medium is a phosphate buffered saline solution, pH 7.4, 10mM, the substrate is added to a final concentration of 485 mmol/L; when the reaction medium was a mixture of 10mM phosphate buffered saline and organic solvent at pH 7.4, the substrate was added to a final concentration of 533 mmol/L.

(I) technical field

The invention relates to preparation of (R) -2-hydroxy-4-phenyl ethyl butyrate, in particular to preparation of (R) -2-hydroxy-4-phenyl ethyl butyrate by using polyacrylic acid polymer as a carrier to co-immobilize glucose dehydrogenase and carbonyl reductase derived from Saccharomyces cerevisiae CGMCC No.3361 and applying the carbonyl reductase to asymmetric reduction of 2-oxo-4-phenyl ethyl butyrate.

(II) background of the invention

(R) -ethyl 2-hydroxy-4-phenylbutyrate (R) -2-hydroxy-4-phenylbutyrate, R-HPBE, CAS number 90315-82-5, molecular formula C₁₂H₁₆O₃208.25, density of 1.075g/mL, boiling point of 212 deg.C, is insoluble in water and soluble in organic solvents. R-HPBE is a key chiral intermediate for synthesizing pril drugs, and has no alternative effect in synthesizing Angiotensin Converting Enzyme Inhibitors (ACEIs). Because of the importance of R-HPBE in producing pril drugs for treating hypertension and cardiovascular diseases, the economic and efficient preparation method of R-HPBE is one of the hot spots of domestic and foreign research. R-HPBE can be prepared by asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE).

The hypertension is a chronic disease which increases arterial pressure due to vascular neuromodulation disorder, and with the improvement of living standard, middle-aged and old people become frequent people of the disease, the hypertension affects vascular fragility, and can cause the problems of vascular rupture and the like in excess, and cardiovascular and cerebrovascular diseases, kidney diseases, eyeground diseases, nervous system diseases and the like are caused, more seriously, the hypertension is closely associated with diseases such as hyperglycemia, high blood viscosity, hyperlipidemia and the like, so that chronic damage to various tissues and organs of the whole body is caused, the function of organs of the body is lost, and the life and health of people are seriously threatened. Lisinopril, quinapril, ramipril, cilazapril and other pril medicines block the generation of angiotensin II by cutting off a renin-angiotensin-aldosterone system to dilate blood vessels and reduce blood pressure, and are safe and effective antihypertensives. Because of the importance of R-HPBE in the synthesis of the drugs, the preparation method of R-HPBE draws much attention at home and abroad, and people continuously strive for the research and development of a new preparation technical route of R-HPBE in recent decades so as to obtain a green synthesis process with high economic and atom utilization rate.

The current methods for synthesizing R-HPBE can be divided into biological methods and chemical methods, and both methods comprise resolution methods and synthesis methods. The chemical resolution method is to resolve the corresponding racemate 2-hydroxy-4-phenylbutyric acid and esterify the racemate into R-HPBE, the resolving agent is a chiral organic amine compound, and the chemical resolution method has the following problems: the optically pure phenethylamine derivative is used as a resolving agent, and the resolving agent has low practical value due to high price; while the resolution yield of the chloramphenicol intermediate is low (less than 50%). The traditional chemical synthesis method has the disadvantages of multiple steps, expensive noble metal catalyst, serious environmental pollution, low enantiomeric excess value of the product, high hydrogen pressure required by catalytic reaction and high requirement on equipment. The biological method is one of the most effective methods for synthesizing R-HPBE at present due to the characteristics of high optical selectivity, mild reaction conditions, environmental friendliness and the like. The theoretical yield of chiral R-HPBE prepared by splitting raceme in the biological resolution method is only 50%, and the product is a mixture and is not easy to separate, so that the method is inferior to the R-HPBE prepared by the biological synthesis method.

The biological synthesis method comprises a whole cell asymmetric reduction method and a reductase conversion method. Biocatalysis using whole cell microorganisms or isolated enzymes as biocatalysts has several advantages and disadvantages. Whole-cell biocatalysts are readily available, relatively inexpensive and more stable, but their catalytic reactions are affected by other metabolic reactions in the cell that produce byproducts, thereby affecting the isolation and purification of the desired product. The isolated enzyme, although expensive, is highly catalytic efficient and highly selective. The product obtained by enzymatic catalysis is convenient for isolation and purification. Carbonyl reductases (CBR) for the asymmetric reduction of carbonyl compounds to prepare chiral alcohols require a coenzyme as a hydrogen donor. In view of the high cost of coenzymes (such as NADH and NADPH), they must be converted from an oxidized form to a recyclable form. Although the microbial conversion method can realize in-situ regeneration of the coenzyme, the substrate treatment amount is very small and is not enough for large-scale production. If the coenzyme can be directly regenerated in situ in the reaction system, the conversion rate can be greatly improved. Glucose Dehydrogenase (GDH) that contributes to coenzyme regeneration is added to supply continuous coenzyme hydrogen donors for asymmetric reduction reactions, depending on the need for coenzyme regeneration.

Traditional enzyme-catalyzed reactions are carried out in the aqueous phase, while a large number of organic substrates are poorly soluble in water and even unstable in the aqueous phase. If the enzyme catalysis is carried out in a single aqueous phase, the chance of the insoluble organic substrate contacting the enzyme is small, and the organic substrate and the product have toxic action on the enzyme, so that the activity of the enzyme is easily reduced, and the conversion rate is low. A direct approach to this problem can be to use an organic-aqueous two-phase system for biocatalysis. The zymoprotein molecules have both hydrophilic and hydrophobic structures, are easy to aggregate at an oil-water interface and self-assemble to form a structure with specific arrangement so as to realize catalytic reaction of the oil-water interface. In order to improve the conversion rate, people use the biological catalysis of an oil-water two-phase system to synthesize chiral alcohol, enzyme can keep the activity and stability in a water phase, a substrate and a product are dispersed in an organic phase, a catalytic reaction occurs at an oil-water interface, and the chiral alcohol is prepared by interface biological catalysis. Organic phase-aqueous phase biphasic catalysis is an environmentally friendly process that offers the opportunity to homogenize the catalyst and substrate/product into two separate and immiscible phases for easy separation. Although oil-water two-phase systems have many advantages, organic phase has negative effects on the activity and stability of enzymes, so that improvement of the activity and stability of enzymes through molecular modification of enzymes is expected.

Due to the flexibility and functional groups of polymers, polymer-enzyme co-binding structures have attracted attention. Some conventional mixed structure or self-assembly polymers, such as chitosan, agarose, polyacrylamide and poly (propyleneimine), have been widely used in biomedical fields such as protein adsorption, biomolecule immobilization, drug release control, and the like. Among these functional polymers, polyacrylic acid (PAA) is a water-soluble biocompatible polymer due to its dispersibility and capacity characteristics. Using a site-specific attachment strategy, a single enzyme can be conjugated to the PAA end groups. The single enzyme may also be randomly coupled within the PAA backbone such that the amino group on the enzyme chemically reacts with the carboxyl group on the PAA.

According to the invention, high-molecular PAA and two enzyme protein molecules (CBR and GDH) are combined through two different synthesis strategies to realize double-enzyme co-immobilization to form nano high-molecular-double-enzyme complexes (BECs), and the BECs are used for preparing an important chiral key body R-HPBE in the synthesis of an angiotensin converting enzyme inhibitor by asymmetrically converting a prochiral substrate OPBE in an oil-water two-phase system, so that the in-situ regeneration of coenzyme is realized, and the optical purity and the substrate conversion rate of the product are improved to the maximum extent.

The invention firstly co-immobilizes CBR with reducing ability and GDH contributing to coenzyme regeneration on a macromolecular PAA skeleton through rational design. The protein and the polymer have different functionalities, and the protein polymer compound formed by combination has the dual characteristics of the two, so that the application efficiency and the range of the two materials are widened. Due to the addition of the polymer, the protein polymer compound can prolong the circulation half-life of the protein, improve the solubility and increase the stability. The PAA is taken as a framework, the CBR and the GDH are co-immobilized by a covalent bonding method, the enzyme separation and loss in the using process are effectively prevented, the enzyme activity and stability at high temperature are improved, the local concentration of the reaction can be increased by double-enzyme co-immobilization, the multi-step reaction can be rapidly completed in the same reactor, the in-situ regeneration of the coenzyme can be realized in a two-phase system, and the catalytic efficiency is improved. The research work of the invention lays a theoretical foundation for preparing the high-efficiency biocatalyst with coenzyme regeneration capacity, and a new path is opened up for improving the biological conversion rate by preparing the nano polymer-double enzyme compound.

Disclosure of the invention

The invention aims to provide a nanometer polymer-double enzyme compound and application thereof in synthesis of (R) -2-hydroxy-4-phenyl ethyl butyrate, and the invention combines glucose dehydrogenase, carbonyl reductase derived from Saccharomyces cerevisiae CGMCC No.3361 and polyacrylic acid by a universal and modular method to realize double-enzyme co-immobilization to prepare the nanometer polymer-double enzyme compound. Carboxyl in polyacrylic acid PAA skeleton and amino in enzyme molecule are covalently combined to play a role in modifying enzyme protein molecules, so that the activity and stability of the enzyme on an oil-water interface are further improved, and the biotransformation rate is improved.

The technical scheme adopted by the invention is as follows:

the invention provides a nano polymer-double enzyme compound, which is prepared by the following method: mixing and stirring carbonyl reductase (CBR) and Glucose Dehydrogenase (GDH) as double enzymes, polyacrylic acid (PAA) as a carrier, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) as a cross-linking agent, Phosphate Buffer (PB) (10mM, pH 7.4) as a reaction medium in a shaking table with the temperature of 20-40 ℃ (preferably 30 ℃) and the rotational speed of 180-220rpm (preferably 200rpm) for 10-60min (preferably 30min) to obtain a nano polymer-double enzyme compound; the carbonyl reductase is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No. 3361.

Further, the mass ratio of the carbonyl reductase to the glucose dehydrogenase is 1:0.5-1 (preferably 1: 0.7); the mass ratio of the total amount of the carbonyl reductase and the glucose dehydrogenase to the polyacrylic acid is 1: 1-5 (preferably 1: 3.75); the mass ratio of the total amount of the carbonyl reductase and the glucose dehydrogenase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20 (preferably 1: 12).

Further, the nano polymer-double enzyme compound is prepared by the following method: mixing and stirring a polyacrylic acid (PAA) solution, carbonyl reductase and glucose dehydrogenase for 10-60min (preferably 30 ℃, 200rpm and 30min) in a shaking table with the rotation speed of 180-220rpm at the temperature of 20-40 ℃, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10mM phosphate buffer solution with the pH of 7.4, mixing and stirring for 10-60min (preferably 30 ℃, 200rpm and 30min) in a shaking table with the rotation speed of 180-220rpm at the temperature of 20-40 ℃, adding 10mM phosphate buffer solution with the volume of 80-120 times, the pH of 7.4 and a 25kDa dialysis membrane for 3 times and 5h each time to remove unreacted EDC and EDC-urea byproducts, and taking a retentate to obtain a nano polymer-double enzyme complex; the polyacrylic acid (PAA) solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with an equal volume of 10mM, pH 7.4 phosphate buffer.

Further, the nano polymer-double enzyme compound is prepared by the following method: (1) mixing polyacrylic acid solution with 10mM phosphate buffer solution with pH 7.4, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring for 10-60min (preferably 30 ℃, 200rpm and 30min) in a shaking table with 220rpm at 20-40 ℃ and 180-; the mass ratio of the carbonyl reductase to the polyacrylic acid is 1: 2-5 (preferably 1: 3.2); the mass ratio of the carbonyl reductase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20 (preferably 1: 10); the polyacrylic acid solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with equal volume of 10mM phosphate buffer solution with pH of 7.4; (2) mixing polyacrylic acid solution with 10mM phosphate buffer solution with pH 7.4, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring for 10-60min (preferably 30 ℃, 200rpm and 30min) in a shaking table with the speed of 180 and 220rpm at the temperature of 20-40 ℃, adding 10mM of glucose dehydrogenase, mixing and stirring for 1-6h (preferably 30 ℃, 200rpm and 2h) in a shaking table with the speed of 180 and 220rpm at the temperature of 20-40 ℃ and pH 7.4 phosphate buffer solution, adding 10mM of 80-120 times volume of the phosphate buffer solution with pH 7.4 and dialyzing a dialysis membrane with the 25kDa for 3 times, and taking trapped fluid every 5h to obtain glucose dehydrogenase-polyacrylic acid conjugate; the mass ratio of the glucose dehydrogenase to the polyacrylic acid is 1: 2-6 (preferably 1: 4.5); the mass ratio of the glucose dehydrogenase to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1: 10-20 parts of; the polyacrylic acid solution is prepared by mixing 0.02g/mL polyacrylic acid aqueous solution with equal volume of 10mM phosphate buffer solution with pH of 7.4; (3) mixing carbonyl reductase-polyacrylic acid conjugate, glucose dehydrogenase-polyacrylic acid conjugate and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, mixing and stirring in a shaking table at 20-40 ℃ and 180-220rpm for 10-60min (preferably 30 ℃, 200rpm and 30min), adding 10mM of 80-120 times volume, pH 7.4 phosphate buffer solution and a dialysis membrane of 25kDa, dialyzing for 3 times, and taking trapped fluid every time for 5h to obtain a nano polymer-double enzyme complex; the volume ratio of the carbonyl reductase-polyacrylic acid conjugate to the glucose dehydrogenase-polyacrylic acid conjugate is 1:0.5-1.5 (preferably 1: 1); the mass ratio of the total mass of the carbonyl reductase-polyacrylic acid conjugate and the glucose dehydrogenase-polyacrylic acid conjugate to the mass of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is 1:0.5-1 (preferably 1: 0.74).

The invention also provides an application of the nanometer polymer-double enzyme compound in preparing (R) -2-hydroxy-4-phenyl ethyl butyrate (R-HPBE) by asymmetrically reducing 2-oxo-4-phenyl ethyl butyrate (OPBE), the application takes 2-oxo-4-phenyl ethyl butyrate (OPBE) as a substrate, taking phosphate buffer solution with pH of 7.4 and 10mM as a reaction medium, taking d-glucose as an auxiliary substrate, takes NADP as coenzyme, takes nano polymer-double enzyme compound as catalyst to form a conversion system, carrying out reduction reaction for 8-40 h (preferably 35 ℃, 180rpm and 40h) at 20-45 ℃ and 50-250rpm, and separating and purifying reaction liquid to obtain (R) -2-hydroxy-4-phenylbutyric acid ethyl ester; the final concentration of the substrate is 300-700 mmol/L (preferably 485-533 mmol/L); the d-glucose is added to a final concentration of 0.05-0.15M (preferably 0.1M); the NADP is added to a final concentration of 0.03-0.07mM (preferably 0.05 mM); the catalyst is added to a final concentration of 0.1-0.6mg/mL (preferably 0.25mg/mL for 2-S and 0.458mg/mL for 2-P).

Further, the reaction medium is a mixture of a phosphate buffered saline solution with pH 7.4 and 10mM and an organic solvent; the volume ratio of the pH 7.4 and the 10mM phosphate buffer salt solution to the organic solvent is 1-5: 3, preferably 1: 1; the organic solvent is n-hexane, dibutyl phthalate, benzene, toluene or n-heptane, preferably dibutyl phthalate.

Further, when the reaction medium was a phosphate buffered saline solution of pH 7.4, 10mM, the substrate was added to a final concentration of 485 mmol/L; when the reaction medium was a mixture of 10mM phosphate buffered saline and organic solvent at pH 7.4, the substrate was added to a final concentration of 533 mmol/L.

Further, the reaction liquid separation and purification method comprises the following steps: after the reaction is finished, adding equal volume of ethyl acetate into the reaction solution for extraction, drying the extract by using anhydrous sodium sulfate, and volatilizing the ethyl acetate to obtain the R-HPBE.

Compared with the prior art, the invention has the following beneficial effects:

the traditional enzyme catalysis reaction is carried out in an aqueous phase, a large amount of organic substrates are insoluble in water, and if the enzyme catalysis is carried out in a single aqueous phase, the chance of contacting the insoluble organic substrates with the enzyme is small, so the conversion rate is low. In order to improve the conversion rate, people disperse the enzyme in an oil-water two-phase system, the molecular structure of the enzyme contains both hydrophilic groups and hydrophobic groups, and the enzyme molecules are rapidly self-assembled on an oil-water interface in the oil-water two-phase system to realize interface catalysis. In the oil-water interface catalysis process, the three-dimensional space structure of enzyme molecules is different from that in the water phase, so the catalytic activity of the enzyme in the oil-water two-phase is usually lower than that in the water phase. Meanwhile, the contact of the organic solvent and the substrate with the enzyme molecules can also cause the change of the molecular structure of the enzyme, and the full play of the enzyme activity is inhibited. In order to improve the catalytic efficiency and stability of enzyme molecules in oil-water two phases and further improve the production efficiency, the invention uses high-molecular polyacrylic acid to modify and transform enzyme protein molecules. Carboxyl in polyacrylic acid PAA skeleton is covalently combined with amino in enzyme molecule to form high molecular-enzyme complex. In order to further improve the conversion rate, the invention adopts a coenzyme regeneration method, and simultaneously adds the coenzyme in the reaction system, and adds glucose dehydrogenase which is helpful for coenzyme regeneration to promote the regeneration of the coenzyme, thereby providing a continuous coenzyme hydrogen donor for asymmetric reduction reaction. Based on the requirement of coenzyme regeneration, glucose dehydrogenase and carbonyl reductase are combined together on a polyacrylic acid polymer skeleton to form a polymer-double enzyme compound, and the polymer-double enzyme compound is used for asymmetric reduction reaction of carbonyl compounds in oil-water two phases, so that coenzyme regeneration can be realized simultaneously.

TABLE 1 comparison of organic-aqueous two-phase systems with Single aqueous reduction

(IV) description of the drawings

FIG. 1 shows two different synthetic routes of the biopolymer-bisase complex.

FIG. 2 is a transmission electron microscope image of the nano polymer-bisase complex 2-S.

FIG. 3 is a SDS-PAGE (SDS-PAGE) electrophoresis of sodium dodecyl sulfate polyacrylamide gels for enzymes (lanes from left to right for GDH, CBR, GDH-PAA, CBR-PAA, 2-P, 2-S, respectively).

FIG. 4 is a circular dichroism plot (CD) of enzymes (GDH, CBR, GDH-PAA, CBR-PAA, 2-P, 2-S).

FIG. 5 is the asymmetric reduction OPBE reaction mechanism of the nanometer macromolecule-double enzyme complex in the organic-aqueous phase two-phase system.

FIG. 6 is a gas chromatogram, wherein A is an S-HPBE standard, B is an R-HPBE standard, C is an OPBE standard, and D is a sample.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the carbonyl reductase (CBR) used in the embodiment of the invention is from Saccharomyces cerevisiae (CGMCC No. 3361), and the strain is preserved in China general microbiological culture Collection center and is disclosed in the patent application CN 101709271B. The carbonyl reductase (CBR) used in the embodiment of the invention is separated and extracted from Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3361 by adopting a method of 2.3 in the literature (Jiqing, Wujian, Lin, Xugang, Yangliang. purification of carbonyl reductase in Candida tropicalis and research of the enzymological properties of the carbonyl reductase, 2009,23 (1): 92-98.) of college and university: ultrasonic cell disruption of Saccharomyces cerevisiae CGMCC No.3361 cells, ammonium sulfate fractional precipitation, DEAE sepharose FF anion exchange chromatography and Blue sepharose 6FF affinity chromatography, and the specific activity of the obtained CBR is 21.4U/mg.

The Glucose Dehydrogenase (GDH) has an enzyme activity of 30U/mg and is purchased from Shanghai Biotech Co., Ltd.

The PB buffer used in the examples of the present invention is 10mM phosphate buffer, pH 7.4.

Example 1: sequential synthesis of nano-polymer-double enzyme compound 2-S

The synthetic route is shown as A in figure 1, and specifically comprises the following steps:

(1) PAA solution: accurately weighed 0.8mg of polyacrylic acid (PAA, Mv ═ 450000g mol)^-1) A PAA stock solution (40. mu.L) of 0.02g/mL was prepared in 40. mu.L of distilled water, and the pH was adjusted to 7. 40 μ L of PAA stock solution was mixed well with 40 μ L of PB buffer to obtain 80 μ L of 0.01g/mL PAA solution.

(2) 80. mu.L of the above 0.01g/mL PAA solution, 0.124mg of CBR and 0.089mg of GDH were mixed and stirred in a shaker at 200rpm and 30 ℃ for 30 minutes to obtain a PAA mixed enzyme solution.

(3) 2.585mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is added into the PAA mixed enzyme solution in the step (2), then PB buffer solution is added to make the total volume of the solution be 1mL, the solution is crosslinked for 30min under the stirring condition of 30 ℃ and 200rpm, the crosslinked mixture is added into 100mL of PB buffer solution, dialysis is carried out for 3 times and 5h each time by using a 25kDa dialysis membrane to remove unreacted EDC and EDC-urea byproducts, and the retentate is taken to obtain 3mg of nano polymer-double enzyme complex which is recorded as BECs (2-S for short). The final enzyme activity of each enzyme (CBR and GDH) in 2-S was 2.5U (1U is defined as the amount of enzyme that can convert 1umol of substrate at 1 min). The particle size of the prepared 2-S nano polymer-double enzyme complex ranges from 50 nm to 70nm (see A in figure 2). The success of the 2-S synthesis was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectroscopy (J-815; JASCO, Japan) (see FIGS. 3 and 4).

Example 2: parallel synthesis of nano-polymer-double enzyme compound 2-P

Referring to fig. 1B, the parallel method is used to synthesize the nano polymer-double enzyme complex 2-P, specifically:

(1) CBR-PAA conjugate: mu.L of a PAA stock solution (prepared as described in example 1) at 0.02g/mL was mixed with an equal volume of PB buffer, 1.292mg of EDC was added, after mixing and stirring in a shaker at 30 ℃ and 200rpm for 30min, 50. mu.L of CBR solution (prepared by dissolving 0.124mg of CBR in PB buffer) was added dropwise and stirred for 2h, 50mL of PB buffer was added, and dialyzed 3 times for 5h each time against a 25kDa dialysis membrane to obtain 1.8mg of CBR-PAA conjugate.

(2) GDH-PAA conjugates: the CBR in the CBR solution in step (1) was replaced with 0.089mg of GDH, and the other operations were the same, to obtain 1.70mg of GDH-PAA conjugate.

(3) Nano polymer-double enzyme complex: 1.8mg of CBR-PAA conjugate and 1.70mg of GDH-PAA conjugate are mixed, 2.585mg of EDC is added to form a 1mL reaction system, the mixture is mixed and stirred for 30min in a shaker at 30 ℃ and 200rpm, 100mL of PB buffer solution is added, dialysis is carried out for 3 times for 5h each time by using a 25kDa dialysis membrane to remove unreacted EDC and EDC-urea byproducts, and a trapped solution is taken to obtain 5.5mg of nano polymer-double enzyme complex (noted as BECs, abbreviated as 2-P). The final enzyme activity of each enzyme (CBR and GDH) in 2-P was 2.5U. The particle size of the prepared 2-P particles is in the range of 50-70nm (see B in FIG. 2). The success of 2-P synthesis was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectroscopy (see FIGS. 3 and 4).

Example 3: effect of substrate concentration in Single aqueous phase on reduction reaction

Two BECs (3mg 2-S or 5.5mg 2-P) prepared in examples 1 and 2 were added to PB buffer containing OPBE at a final concentration of 0.05mM NADP, 0.1M d-glucose, and 388mM,436mM,485mM,533mM,582mM, and 630mM, respectively, to make up a total reaction system volume of 12 mL. Reducing in a constant temperature shaking table at 35 deg.C and 180rpm for 40h, adding equal volume of ethyl acetate into the reaction solution after reaction, extracting, drying the extract (upper layer) with anhydrous sodium sulfate, and detecting the obtained product by gas chromatography, the results are shown in Table 2.

The conversion of the substrate, defined as the ratio of the concentration of the substrate after conversion to the initial substrate concentration, and the enantiomeric excess of the product R-HPBE were analyzed by Shimadzu GC-2014 gas chromatography. By being equipped with Agilent CP 7502J&W CP-Chirasil-Dex CB chiral column (Machery-Nagel; 25 m.times.0.25 mm.times.0.25 m)m) was analyzed by Gas Chromatography (GC). The injection port, chromatography column and FID detector temperatures were 250, 130 and 250 ℃ respectively. The split ratio is 1: 15. the flow rate was 2 mL/min. The retention times of OPBE, R-HPBE and S-HPBE were 20.898, 27.948 and 28.923 minutes, respectively. The conversion rate (X) of OPBE and the enantiomeric excess value (ee) of HPBE were calculated using the formulas (1) and (2)_p)。

Ms and Mp are the molecular weights of the substrate and product. P represents the mass of the product at the end of the reaction and Q represents the initial mass of the substrate.

C_RAnd C_SRepresents the concentration of R-HPBE and S-HPBE.

TABLE 2 Effect of different substrate concentrations in a Single aqueous phase on the reduction reaction

In this experiment, the effect of OPBE concentration (388- & ltSUB & gt, 630mM) on the reduction reaction was investigated. Table 2 shows that high concentrations of OPBE have some toxic effects on the enzyme, but too low concentrations of OPBE reduce the efficiency of the reaction. During the increase of OPBE concentration from 388mM to 485mM, all substrates were still completely converted to HPBE. At concentrations above 485mM, the conversion started to show a downward trend. When 2-S and 2-P catalyze 533mM OPBE, the conversion rate can reach 94.17% and 87.08%, respectively. When the concentration of OPBE exceeded 485mM, the enantiomeric excess of R-HPBE catalyzed by 2-P was slightly reduced, while the peak (99.9%) was maintained with 2-S as catalyst. Differences in the structure and preparation process of the two types of nanobecs (2-P and 2-S) may lead to differences in the resistance to substrate concentration. Obviously, the most suitable OPBE concentration is 485mM for both 2-S and 2-P.

Example 4: influence of shaking table rotation speed on reduction reaction in single aqueous phase

485mM substrate OPBE at final concentration, d-glucose as co-substrate at final concentration of 0.1M, coenzyme NADP at final concentration of 0.05mM and nanobECs (3mg 2-S or 5.5mg 2-P) were added to a reaction flask containing PB buffer, and the total volume of the reaction system was kept at 12 mL. The reaction was carried out for 40h in a constant temperature (35 ℃) shaker at different rotational speeds (150rpm, 160rpm, 170rpm, 180rpm, 190 rpm). After the reaction was completed, the substrate conversion rate and the enantiomeric excess (%) of R-HPBE were measured by the method of example 3.

TABLE 3 influence of the rotational speed of the shaker on the reduction reaction in a Single aqueous phase

The rotational speed of the shaker will affect the diffusion and distribution of the substrates and products in the reaction system, which will ultimately affect the molar conversion and product configuration. Table 3 shows that when the constant temperature shaker speed is less than 180rpm, the conversion increases with increasing shaker speed, resulting in a corresponding increase in molar conversion, indicating that mass transfer is the primary determinant of the reaction in this case. If the shaker speed is further increased above 180rpm, both the conversion and the enantiomeric excess of R-HPBE will remain unchanged. Thus, for both types of nano-BECs (2-S and 2-P), 180rpm was considered the most suitable shaker speed.

Example 5: influence of organic solvent on reduction reaction in organic-aqueous two-phase system

Two types of nano BECs (3mg 2-S or 5.5mg 2-P) prepared in examples 1 and 2 were dispersed in PB buffer to form 6mL of aqueous phase, and 6mL of organic solvents (n-hexane, dibutyl phthalate, benzene, toluene, n-heptane) of different kinds were added to the aqueous phase to form five two-phase systems in equal volumes, and 533mM OPBE was added to a final concentration of 0.05mM OPBENADP, final concentration 0.1M d-glucose, constituted 12mL of the total volume of the reaction system, was reduced in a shaker at 35 ℃ and 120rpm for 40 hours. After the reaction, the mixture was added with ethyl acetate of the same volume and mixed well, and centrifuged at 8000rpm for 10 minutes to separate the organic phase. The organic phase is passed through anhydrous Na₂SO₄The substrate conversion and the enantiomeric excess (%) of R-HPBE were determined by drying in accordance with the method of example 3, and the results are shown in Table 4.

TABLE 4 influence of organic solvent on the reduction reaction in organic-aqueous two-phase System

The selection of a suitable organic solvent in situ extractant is very important for organic-aqueous biphasic systems. This example discusses the biocatalysis in a biphasic system consisting of different organic solvents and PB, the results of which are shown in table 4. The logP (hydrophobic constant) of an organic solvent is an important constant for evaluating its effect on an enzyme catalyst. It is the logarithm of the solvent distribution constant in the n-butanol/water system. It was found experimentally that when dibutyl phthalate (1ogP > 5) was used as the organic phase, the molar conversion and enantiomeric excess of R-HPBE were higher than for the other four organic solvents. The reason for the low conversion rate of other organic solvents (n-hexane, benzene, toluene, n-heptane) used as the organic phase may be that their poisoning effect on the enzyme causes the enzyme to lose most of its activity, thereby affecting the catalytic performance of the enzyme. According to the experimental results, dibutyl phthalate having good biocompatibility and extraction properties was selected as the best organic phase for the following studies.

Example 6: influence of phase volume ratio on reduction reaction in organic-aqueous two-phase System

The nanobECs (3mg 2-S or 5.5mg 2-P) prepared in examples 1 and 2 were dispersed in different volumes (3, 4.8, 6, 6.8, 7.5mL) of PB buffer to form aqueous phases. Thereafter, different volumes (9, 7.2, 6, 5.2, 4.5mL) of dibutyl phthalate were added so that the volume ratio of the organic phase to the aqueous phase was 1:3. 2: 3. 3: 3. 4: 3. 5: 3 forming a two-phase system, addingThe concentration of 533mM OPBE, the final concentration of 0.05mM NADP, and the final concentration of 0.1M d-glucose made up a total volume of 12mL of the reaction system. The above reaction flask having different phase volume ratios was placed in a constant temperature oscillator (35 ℃, 120rpm) to react for 40 hours, and then extracted with an equal volume of ethyl acetate. The organic phase is passed through anhydrous Na₂SO₄After drying, the substrate conversion and the enantiomeric excess of R-HPBE were measured by the method of example 3, and the results are shown in Table 5.

TABLE 5 influence of phase volume ratio on reduction reaction in organic-aqueous two-phase System

In general, the volume ratio of organic solvent and water has a greater effect than the enzyme-catalyzed reaction. Too large a ratio will result in severe enzyme inactivation. However, when the phase volume ratio is too low, the object of increasing the substrate processing ability and reducing the inhibition of the enzyme-catalyzed reaction by the substrate and the product cannot be achieved. This example investigated the effect of the volume ratio of organic solvent to PB (0% -62.5%) in a two-phase dibutyl phthalate-PB reaction system on the molar conversion and the enantiomeric excess of R-HPBE. As can be seen from Table 5, the phase volume ratio (Vo/Va) has a significant effect on the substrate processing ability, while the enantiomeric excess of R-HPBE has little effect. As Vo/Va increases over a range (< 1), the molar conversion shows a tendency to increase slowly and the conversion is at a higher level. As the proportion of organic solvent continues to increase, the conversion drops severely. Therefore, a 50% organic solvent ratio (Vo/Va of 1:1) was selected as an optimum volume ratio for further study.

Example 7: influence of reaction temperature on reduction reaction in organic-aqueous two-phase system

The nano BECs (3mg 2-S or 5.5mg 2-P) prepared in examples 1 and 2 were dispersed in 12mL of a two-phase system in which dibutyl phthalate and PB were present in a volume ratio of 1:1, and 533mM OPBE, 0.05mM NADP and 0.1M d-glucose were added to make up a total volume of 12mL, and the mixture was placed in a constant temperature shaker (120rpm) at 20 ℃ and 25 ℃ respectivelyReacted at 30 ℃, 35 ℃ and 40 ℃ for 40h, then extracted with an equal volume of ethyl acetate and centrifuged (8000rpm, 10min) to obtain a separated aqueous and organic phase. The organic phase is passed through anhydrous Na₂SO₄After drying, the substrate conversion and the enantiomeric excess of R-HPBE were measured by the method of example 3, and the results are shown in Table 6.

TABLE 6 influence of reaction temperature on the reduction reaction in an organic-aqueous two-phase System

In enzymatic reactions, temperature has varying degrees of influence on the activity and stereoselectivity of biocatalysts. At the optimum reaction temperature, the biocatalyst may exhibit high catalytic activity. This experiment investigated the change in the conversion of OPBE and the enantiomeric excess of R-HPBE in the range of 20-40 ℃. Table 6 shows that the conversion increases significantly to a stable maximum (100%) when the temperature is increased from 20 ℃ to 35 ℃. As the temperature is further increased, the conversion begins to drop. The law of change of the conversion rate influenced by the temperature is simultaneously applicable to the reduction reaction catalyzed by 2-S and 2-P. This law of change may be due to the fact that excessive temperatures destroy the spatial structure of the enzyme, and a decrease in the activity of the enzyme leads to a decrease in the conversion rate. The enantiomeric excess value of the R-HPBE does not change significantly with temperature. Table 6 shows that when the reaction temperature was 35 ℃, the molar conversion and the enantiomeric excess of R-HPBE reached 100% and 99.9%, respectively. Therefore, the optimum temperature for the reduction reaction using 2-S or 2-P as a catalyst is 35 ℃.

Example 8: effect of substrate concentration on reduction reactions in organic-aqueous two-phase systems

To 12mL of a two-phase system in which dibutyl phthalate and PB buffer were present in a volume ratio of 1:1, 2-S was added in an amount of 3mg or 2-P was added in an amount of 5.5mg, respectively, to the two-phase system, and then 388mM,436mM,485mM,533mM,582mM, and 630mM OPBE, respectively, at a final concentration of 0.05mM NADP, and 0.1M d-glucose were added, and then six reaction flasks were placed in a shaker (35 ℃, 120rpm) and reacted for 40 hours. At the end of the reduction reaction, the reaction solution was treated with ethyl acetate of equal volumeAnd (4) ester extraction. Centrifugation (8000rpm, 10min) was carried out to obtain a separated aqueous phase and an organic phase. The organic phase is passed through anhydrous Na₂SO₄After drying, the substrate conversion and the enantiomeric excess of R-HPBE were measured by the method of example 3, and the results are shown in Table 7.

TABLE 7 influence of substrate concentration on the reduction reaction in organic-aqueous two-phase System

The substrate concentration will affect the catalytic efficiency of the BECs at the interface between the organic and aqueous phases. Without the use of substrate feed, the present study examined the effect of different initial substrate concentrations (388- & ltSUB- & gt, 630mM) on asymmetric reduction in a biphasic system with 2-S and 2-P as biocatalysts. Table 7 shows that the conversion is always stabilized at the highest value (100%) when the substrate concentration is increased from 388mM to 533 mM. If the substrate concentration is further increased, the conversion will decrease dramatically. High substrate concentrations (>533mM) may lead to excessive accumulation of substrate in the aqueous phase, which in turn inhibits the reaction. The enantiomeric excess of R-HPBE was found to be hardly affected by the substrate concentration. Thus, for the reduction reaction with 2-P and 2-S as catalysts in the dibutyl phthalate-PB (1: 1) biphasic system, the most suitable substrate concentration is 533 mM.

Example 9: influence of table rotation speed on reduction reaction in organic-aqueous two-phase system

To 12mL of a two-phase system in which dibutyl phthalate and PB buffer were present in a volume ratio of 1:1, 2-S in an amount of 3mg or 2-P in an amount of 5.5mg were added, and 533mM OPBE was added thereto in a final concentration of 0.05mM NADP and in a final concentration of 0.1M d-glucose, and the mixture was placed in a shaker (80rpm, 100rpm, 120rpm, 150rpm, 180rpm) and reacted at 35 ℃ for 40 hours. At the end of the reduction reaction, the reaction solution was extracted with an equal volume of ethyl acetate. Centrifugation (8000rpm, 10min) was carried out to obtain a separated aqueous phase and an organic phase. The organic phase is passed through anhydrous Na₂SO₄After drying, the substrate conversion and the enantiomeric excess of R-HPBE were measured by the method of example 3, and the results are shown in Table 8.

TABLE 8 influence of the rotational speed of the rocking bed on the reduction reaction in an organic-aqueous two-phase System

The suitable rotation speed of the constant temperature oscillator can ensure that the substrate and the product are more fully dissolved in the dibutyl phthalate and improve the material transfer efficiency. In this experiment, the influence of the table rotation speed in the range of 80-180rpm on the conversion and the enantiomeric excess of R-HPBE was investigated, with other conditions being kept constant. In the range of 80-120rpm, the conversion increased until a maximum (100%) was reached and then stabilized with increasing speed. When the oscillator speed is below 120rpm, insufficient contact between the biocatalyst nanobECs (2-S and 2-P) and the organic substrate at the oil-water interface may result. Using 2-P or 2-S as catalyst, the optimum oscillator speed was 120 rpm. 2-P or 2-S can obtain the maximum catalytic activity in the reaction system at 120rpm (see FIG. 6).

Example 10: exploration of coenzyme regeneration capacity in organic-aqueous two-phase system

To a 12mL two-phase system having a dibutyl phthalate/PB buffer volume ratio of 1:1, 1.7mg of GDH-PAA, 1.8mg of CBR-PAA, and nano-BECs (3mg of 2-S or 5.5mg of 2-P) were added, and after adding 533mM OPBE at a final concentration, 0.05mM NADP at a final concentration, and 0.1M d-glucose at a final concentration, the mixture was placed in a shaker (35 ℃, 120rpm), and reacted for 40 hours. At the end of the reduction reaction, the reaction solution was extracted with an equal volume of ethyl acetate. Centrifugation (8000rpm, 10min) was carried out to obtain a separated aqueous phase and an organic phase. The organic phase is passed through anhydrous Na₂SO₄After drying, the substrate conversion and the enantiomeric excess of R-HPBE were measured by the method of example 3, and the results are shown in Table 9.

TABLE 9 coenzyme regeneration Capacity study in organic-aqueous two-phase System

CBR-PAA, GDH-PAA and the dual enzyme-PAA conjugates (2-S and 2-P) were used as catalysts in dibutyl phthalate-PB (1: 1) biphasic system to study the regeneration capacity of coenzymes. The product R-HPBE was not detected in the reaction system using GDH-PAA as a catalyst. Furthermore, the amount of R-HPBE obtained in the reaction with CBR-PAA as a catalyst (349mM) was significantly lower than that obtained in the reaction with the double enzyme-PAA conjugate 2-S or 2-P) as a catalyst (533 mM). The results show a 34.52% increase in the total conversion of co-immobilization of CBR and GDH on polymeric PAA compared to CBR-PAA (table 2). In conjunction with the analysis of the reaction mechanism (FIG. 5), the nanobECs (2-S or 2-P) have the ability of coenzyme regeneration, which contributes to the improvement of the conversion rate.