CN111686809A - Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method and application thereof - Google Patents
Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method and application thereof Download PDFInfo
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- CN111686809A CN111686809A CN202010570195.9A CN202010570195A CN111686809A CN 111686809 A CN111686809 A CN 111686809A CN 202010570195 A CN202010570195 A CN 202010570195A CN 111686809 A CN111686809 A CN 111686809A
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/06—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing polymers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
- C07C67/31—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of functional groups containing oxygen only in singly bound form
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
- B01J2231/60—Reduction reactions, e.g. hydrogenation
- B01J2231/64—Reductions in general of organic substrates, e.g. hydride reductions or hydrogenations
- B01J2231/641—Hydrogenation of organic substrates, i.e. H2 or H-transfer hydrogenations, e.g. Fischer-Tropsch processes
- B01J2231/643—Hydrogenation of organic substrates, i.e. H2 or H-transfer hydrogenations, e.g. Fischer-Tropsch processes of R2C=O or R2C=NR (R= C, H)
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Abstract
The invention belongs to the technical field of biological pharmacy, and particularly discloses a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, and a preparation method and application thereof. The invention co-embeds carbonyl reductase and isopropanol dehydrogenase in a solid phase carrier obtained by polyvinyl alcohol and polyethylene glycol, and the obtained co-immobilized enzyme catalyst can realize the high-efficiency and high-stereoselectivity reduction generation of 3-carbonyl-5-hexenoic acid ester by catalyzing the carbonyl reductase and circulating the isopropanol dehydrogenase to coenzyme NADPH (the invention)R) -3-hydroxy-5-hexenoic acid ester. The catalyst can be used for preparing (by taking 3-carbonyl-5-hexenoate as a substrate)R) In the (E) -3-hydroxy-5-hexenoic acid ester, the co-supported catalyst prepared by the method has high catalytic efficiency, good stability, reusability, simple process and finished productThe cost is low, and the method has excellent practical industrial application value.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a co-immobilized enzyme catalyst, a preparation method thereof and a method for synthesizing (A)R) -3-hydroxy-5-hexenoic acid ester.
Background
（R) The (E) -3-hydroxy-5-hexenoic acid ester is an important chiral compound which is widely applied to the synthesis of pharmaceutical and chemical intermediates including statin hypolipidemic drugs, and the structural formula of the compound is shown as the following figure (I), wherein R is C1-C8Alkyl or cycloalkyl, mono-or poly-substituted aryl or aralkyl.
As the main route for the synthesis of the compounds (I) and their structural analogues at present, the chemical methods have disadvantages such as the often use of extreme reaction conditions, including-20oC to-50oCryogenic temperature (US patent US 6355822) and high hydrogen pressure (european patent EP 1176135). These harsh reaction conditions severely limit the industrial prospects of chemical processes for the preparation of such compounds.
The team reported that the reduction of Compound (II) by the enzyme carbonyl reductase was highly efficient and highly stereoselective: (>99%ee) Generation of (R) -3-hydroxy-5-hexenoic acid ester (I) (chinese patent application CN107119081A, US patent US 10526622). Compared with the traditional chemical method, the method has the advantages of mild reaction conditions and high reaction yield, and has good industrial application value. However, in this report, both carbonyl reductase for catalyzing the reaction and isopropanol dehydrogenase for coenzyme regeneration are crude enzyme solutions, which makes the post-treatment of the reaction complicated, and increases the industrial production cost because the enzyme catalyst is difficult to recover and reuse.
Disclosure of Invention
The invention aims to provide a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst with high catalytic efficiency and good stability, a preparation method thereof and a synthesis process thereofR) -3-hydroxy-5-hexenoic acid ester.
The invention provides a preparation method of a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which comprises the following specific steps:
step 1: preparing aqueous solution of polyvinyl alcohol and polyethylene glycol with a certain concentration, heating for dissolving, and cooling to below 50 ℃;
step 2: adding a certain proportion of carbonyl reductase crude enzyme liquid and isopropanol dehydrogenase crude enzyme liquid (the preparation method of the crude enzyme liquid is shown in our earlier Chinese patent application CN 107119081A) into the solution obtained in the step 1, and uniformly mixing;
and step 3: after mixing, the solution is dripped on a polyethylene film by a syringe, and then the polyethylene film is dried for a certain time in a 35-40 ℃ blast oven to obtain the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst which is stored at 4 ℃ for later use.
Wherein, the amino acid sequence of the carbonyl reductase is shown as SEQ ID NO.1, and the amino acid sequence of the isopropanol dehydrogenase is shown as SEQ ID NO. 2.
In step 1 of the present invention, preferably, the mass ratio of the polyvinyl alcohol to the polyethylene glycol is 5:3 to 5:1, and more preferably 5:3 to 5: 2.
In step 2 of the present invention, the initial concentrations of the crude enzyme solution of carbonyl reductase and the crude enzyme solution of isopropanol dehydrogenase are preferably 10% to 30% (w/v), and more preferably 15% to 20% (w/v).
In step 2 of the present invention, preferably, the volume ratio of the carbonyl reductase crude enzyme solution, the isopropanol dehydrogenase crude enzyme solution and the polyvinyl alcohol/polyethylene glycol aqueous solution is 2: 1: 5-2: 1: 10, more preferably 2: 1: 5-2: 1: 7.
in step 3 of the invention, the drying time is 0.5-1 h.
The invention co-embeds carbonyl reductase and isopropanol dehydrogenase in a solid phase carrier obtained from polyvinyl alcohol and polyethylene glycol, and the obtained co-immobilized enzyme catalyst realizes the high-efficiency and high-stereoselectivity reduction generation of 3-carbonyl-5-hexenoic acid ester (II) through the catalysis of the carbonyl reductase and the circulation of the isopropanol dehydrogenase to coenzyme NADPHR) -3-hydroxy-5-hexenoic acid ester (I).
The co-immobilized enzyme catalyst prepared by the invention can be used for preparing (II) by taking 3-carbonyl-5-hexenoic acid ester (II) as a substrateR) -3-hydroxyThe method comprises the following specific steps of (I) 5-hexenoic acid ester:
co-immobilized enzyme catalyst, phosphate buffer solution, isopropanol, coenzyme NADP+Mixing with 3-carbonyl-5-hexenoic acid ester (II) as substrate, and performing catalytic reaction at pH 6-9 and reaction temperature 15-35oC, preparing (A)R) -3-hydroxy-5-hexenoic acid ester (I);
the reaction formula of the invention is as follows:
in the formula, R is C1-C8Alkyl or cycloalkyl, mono-or poly-substituted aryl or aralkyl.
As shown in the reaction formula, in the reaction, the substrate 3-carbonyl-5-hexenoic acid ester (II) is subjected to reduction reaction under the catalysis of the co-immobilized enzyme catalyst to generate a product (I)R) And (3) -3-hydroxy-5-hexenoic acid ester (I), and after the reaction is finished, separating and purifying the reaction liquid to obtain the target product.
Preferably, the substrate is present in the initial reaction system at a concentration of 1% to 30% (w/v), more preferably 10% to 20% by mass.
Preferably, the percentage concentration of isopropanol in the initial reaction system is between 5% and 25% (v/v), more preferably between 10% and 20%.
Preferably, the co-immobilized enzyme catalyst is used in an amount of 20% to 100% (w/w), more preferably 50% to 100%, of the mass of the substrate.
Preferably, the coenzyme NADP+The dosage of the compound is 0.003-0.01% (w/w) of the dosage of the substrate.
Preferably, the reaction temperature is 25 to 30 DEG CoC；
Preferably, the pH of the reaction solution is 6.5 to 7.5.
After the reaction is finished, carrying out post-treatment on the reaction solution to obtain a finished product, wherein the post-treatment comprises the following steps: filtering and separating the immobilized enzyme catalyst, and extracting the filtrate for 3 times by using ethyl acetate; and combining organic layers, washing with water and saturated saline solution respectively, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to dryness to obtain a finished product.
Compared with the prior art, the catalyst has high catalytic efficiency, good stability and reusability, and shows more excellent stability and catalytic efficiency by comparing the co-immobilized enzyme catalyst with free enzyme in the aspects of temperature stability, pH stability, reusability times and the like. The method has the advantages of simple process, low cost and great industrial application value.
Drawings
FIG. 1 is a graph showing the temperature stability of isopropanol dehydrogenase free enzyme and co-immobilized enzyme catalyst.
FIG. 2 is a graph of temperature stability of carbonyl reductase free enzyme and co-immobilized enzyme catalyst.
FIG. 3 is a graph showing pH stability curves of isopropanol dehydrogenase free enzyme and co-immobilized enzyme catalyst.
FIG. 4 is a pH stability curve for carbonyl reductase free enzyme and co-immobilized enzyme catalyst.
FIG. 5 is a graph showing the storage stability of isopropanol dehydrogenase-free enzyme, carbonyl reductase-free enzyme, and co-immobilized enzyme catalyst.
FIG. 6 is a study of the reusability of the co-supported catalyst.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.
Example 1 preparation of carbonyl reductase/Isopropanol dehydrogenase Co-immobilized catalyst
Weighing 5g of polyvinyl alcohol, 3g of polyethylene glycol and 35mL of water, placing the mixture in a reaction bottle, heating the mixture until the solution is clear, cooling the solution to below 50 ℃, adding 10mL of carbonyl reductase crude enzyme solution (15% w/v) and 5mL of isopropanol dehydrogenase crude enzyme solution (15% w/v), and uniformly mixing. After mixing, the solution is dripped on a polyethylene film by a syringe, and then the polyethylene film is dried in a forced air oven at the temperature of 35-40 ℃ for 1h to obtain the carbonyl reductase/isopropanol dehydrogenase co-supported catalyst which is stored at the temperature of 4 ℃ for later use.
Example 2 study of the thermal stability of the carbonyl reductase/Isopropanol dehydrogenase Co-immobilized catalyst
The carbonyl reductase free enzyme (crude enzyme solution), the isopropanol dehydrogenase free enzyme (crude enzyme solution) and the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in example 1 are taken, heat preservation is carried out for 30min at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, then the enzyme activity is respectively measured, the highest activity is 100%, the measurement is carried out for three times in parallel, and corresponding temperature stability curves are made, as shown in figures 1 and 2.
The enzyme activity determination method of carbonyl reductase comprises the following steps: adding a certain amount of carbonyl reductase free enzyme or immobilized enzyme into phosphate buffer solution (100 mM, pH 7.5) containing substrate (10 mM) and NADPH (0.24 mM), shaking for 1min, centrifuging, collecting supernatant, measuring absorbance change at 340 nm, and calculating enzyme activity per gram of enzyme.
The method for measuring the enzyme activity of the isopropanol dehydrogenase comprises the following steps: adding a certain amount of isopropanol dehydrogenase free enzyme or immobilized enzyme into the mixture containing isopropanol (2% v/v) and NADP+(0.4 mM) in phosphate buffer solution (100 mM, pH 7.5), shaking for 1min, centrifuging, collecting supernatant, measuring absorbance change at 340 nm, and calculating enzyme activity per gram of enzyme.
As can be seen from FIGS. 1 and 2, the isopropanol dehydrogenase activity of the immobilized enzyme was higher than that of the free enzyme, but the activity of the immobilized enzyme was 20% at 80 ℃ and the temperature stability of the immobilized enzyme was higher than that of the free enzyme at 40 ℃ to 80 ℃. For carbonyl reductases, the immobilized enzyme has a higher temperature stability than the free enzyme at a temperature in the range of 30-70 ℃. The results show that: in a high-temperature environment, the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is more stable than free enzymes of isopropanol dehydrogenase and carbonyl reductase, and can better keep catalytic activity.
Example 3 pH stability study of carbonyl reductase/Isopropanol dehydrogenase Co-immobilized catalyst
Adding carbonyl reductase free enzyme, isopropanol dehydrogenase free enzyme and the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in the example 1 into buffer solutions with pH values of 4, 5, 6, 7 and 8, incubating for 5min under the condition of water bath at 30 ℃, then respectively measuring the enzyme activity, taking the highest activity as 100%, and performing parallel measurement for three times to obtain corresponding pH stability curves, as shown in FIGS. 3 and 4.
As is clear from fig. 3 and 4, in the case of isopropanol dehydrogenase, both the immobilized enzyme and the free enzyme lost activity at pH =4, and the pH stability of the immobilized enzyme was improved compared to that of the free enzyme at pH =5 to 8. For carbonyl reductase, the pH stability of immobilized enzyme is reduced compared to free enzyme under acidic conditions; however, under alkaline conditions, the pH stability of the immobilized enzyme is higher than that of the free enzyme. The results show that: under alkaline conditions, the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is more stable than free enzymes of isopropanol dehydrogenase and carbonyl reductase, and can better maintain catalytic activity.
Example 4 study of storage stability of carbonyl reductase/Isopropanol dehydrogenase Co-immobilized catalyst
The carbonyl reductase free enzyme, the isopropanol dehydrogenase free enzyme, and the carbonyl reductase/isopropanol dehydrogenase co-supported catalyst prepared in example 1 were allowed to stand at 20 ℃ for 12 days, and the enzyme activities were measured for 4 days and 12 days, respectively, using the initial activity as 100%, to obtain a graph 5.
As can be seen from FIG. 5, the stability of the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst was higher than that of the free enzyme after 12 days of storage. Wherein the enzyme activity of the carbonyl reductase of the co-immobilized catalyst is basically not changed, and the enzyme activity of the isopropanol dehydrogenase is also kept above 85%. The activities of both the carbonyl reductase free enzyme and the isopropanol dehydrogenase free enzyme are reduced to below 80%, wherein the activity of the isopropanol dehydrogenase free enzyme is only 60%. The results show that: the storage stability of the immobilized enzyme at room temperature is more excellent than that of the free enzyme, and the immobilized enzyme can be stored for a long time.
Example 5 study of reusability of carbonyl reductase/Isopropanol dehydrogenase Co-immobilized catalyst
The carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (100 mg) prepared in example 1 was added to the substrates, i.e., tert-butyl 3-carbonyl-5-hexenoate (100 mg), isopropanol (0.1 mL), and NADP+(0.01 mg), phosphate buffer (50 mM, pH = 7)0) (0.4 mL) was reacted in a constant temperature shaking table for 10 hours (30 ℃ C., 200 rpm), and after completion of the reaction, the conversion of the substrate was measured by GC-MS, and then the co-immobilized enzyme catalyst was recovered by filtration and washed clean. The co-immobilized enzyme catalyst was used repeatedly 18 times in this manner, and the conversion was measured to prepare a reuse performance chart (FIG. 6).
As can be seen from fig. 6, the substrate conversion rate of the immobilized enzyme was substantially stable (> 98%) after 10 times of repeated use; after 18 times of repeated use, the substrate conversion rate is still kept above 85%. The results show that: the enzyme is embedded in the immobilized carrier stably and is not easy to fall off, the enzyme activity is kept stable, and the enzyme can be used repeatedly.
Example 6 production of asymmetric reduction catalyzed by carbonyl reductase/isopropanol dehydrogenase Co-immobilized catalyst: (R) -3-hydroxy-5-hexenoic acid tert-butyl ester (gram-grade)
The carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (1 g) prepared in example 1 was added with the substrates 3-carbonyl-5-hexenoic acid tert-butyl ester (1 g), isopropanol (1 mL), NADP+(0.1 mg) and phosphate buffer (50 mM, pH = 7.0) (4 mL) were reacted in a constant temperature shaking table for 10 hours (30 ℃ C., 200 rpm), and after completion of the reaction, the co-immobilized enzyme catalyst was recovered by filtration. Extracting the filtrate with ethyl acetate for 3 times, mixing organic layers, washing with water and saturated saline solution respectively, drying with anhydrous sodium sulfate, concentrating under reduced pressure to dryness to obtain 0.91 g (yield 90%,eevalue 99.7%).1H NMR (CDCl3, 400 MHz): /ppm 5.82 (m,1H), 5.06 (d,J= 6.4 Hz, 1H), 5.02 (s, 1H), 3.97 (m, 1H),2.40-2.14 (m, 4H)，1.39 (s, 9H)。
Example 7 production of asymmetric reduction catalyzed by carbonyl reductase/isopropanol dehydrogenase Co-immobilized catalyst: (R) -3-hydroxy-5-hexenoic acid methyl ester (gram-grade)
The carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (1 g) prepared in example 1 was added with the substrates methyl 3-carbonyl-5-hexenoate (1 g), isopropanol (0.8 mL), and NADP+(0.07 mg), phosphate buffer (50 mM, pH = 7.0) (4 mL), in a constant temperature shaking table for 12h (30 ℃; 4 mL),200 rpm), after the reaction was completed, the co-immobilized enzyme catalyst was recovered by filtration. Extracting the filtrate with ethyl acetate for 3 times, mixing organic layers, washing with water and saturated saline solution respectively, drying with anhydrous sodium sulfate, concentrating under reduced pressure to dryness to obtain 0.90 g (yield 89%,eevalue 99.8%).1H NMR (CDCl3, 400 MHz): /ppm5.81 (m, 1H), 5.14 (d,J= 4.6 Hz, 1H), 5.10 (s, 1H), 4.07 (m, 1H),3.70 (s,3H)，2.48-2.24 (m, 4H)。
The above description is only for the purpose of illustrating the present invention and the technical idea and features thereof, and it is intended to enable those skilled in the art to understand the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. Any modification, equivalent replacement, improvement and the like made in accordance with the spirit of the present invention shall be covered within the protection scope of the present invention.
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Claims (10)
1. A preparation method of a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is characterized by comprising the following specific steps:
step 1: preparing aqueous solution of polyvinyl alcohol and polyethylene glycol, heating for dissolving, and cooling to below 50 ℃;
step 2: adding the crude enzyme solution of carbonyl reductase and the crude enzyme solution of isopropanol dehydrogenase into the solution obtained in the step 1, and uniformly mixing;
and step 3: after mixing, the solution is dripped on a polyethylene film by an injector, and then the polyethylene film is dried in a blast oven at the temperature of 35-40 ℃ to obtain the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst which is stored for later use.
2. The preparation method according to claim 1, wherein the mass ratio of the polyvinyl alcohol to the polyethylene glycol in step 1 is 5:3 to 5: 1.
3. The method according to claim 1, wherein the initial concentrations of the crude enzyme solution of carbonyl reductase and the crude enzyme solution of isopropanol dehydrogenase used in step 2 are both 10% to 30% (w/v).
4. The method according to claim 1, wherein the volume ratio of the crude enzyme solution of carbonyl reductase, the crude enzyme solution of isopropanol dehydrogenase and the aqueous solution of polyvinyl alcohol/polyethylene glycol used in step 2 is 2: 1: 5-2: 1: 10.
5. the method according to claim 1, wherein the drying time in step 3 is 0.5 to 1 hour.
6. A carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst obtained by the production method according to any one of claims 1 to 5.
7. Preparation of the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst of claim 6R) -3-hydroxy-5-hexenoic acid ester.
8. The application of claim 7, comprising the following steps:
co-immobilized enzyme catalyst, phosphate buffer solution, isopropanol, coenzyme NADP+Mixing with 3-carbonyl-5-hexenoic acid ester (II) as substrate, and performing catalytic reaction at pH 6-9 and reaction temperature 15-35oC, preparing (A)R) -3-hydroxy-5-hexenoic acid ester (I);
the reaction formula is as follows:
in the formula, R is C1-C8Alkyl or cycloalkyl, mono-or poly-substituted aryl or aralkyl.
9. The use according to claim 8, wherein the substrate is present in the initial reaction system at a concentration of 1% to 30% by mass (w/v); the percent concentration of isopropanol is 5% to 25% (v/v).
10. The use according to claim 8, wherein the co-supported catalyst is used in an amount of 20-100% (w/w) of the mass of the substrate; the coenzyme NADP+The dosage of the compound is 0.003-0.01% (w/w) of the dosage of the substrate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375801A (en) * | 2020-10-22 | 2021-02-19 | 复旦大学 | Micro-reaction system and method for continuously preparing (R) -3-hydroxy-5-hexenoic acid ester by using same |
| CN112501235A (en) * | 2020-11-19 | 2021-03-16 | 华东师范大学 | Method for preparing breynolone by using monoalcohol ketone acetate |
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| US11913055B2 (en) | 2020-10-22 | 2024-02-27 | Fudan University | Continuous flow method for preparing (R)-3-hydroxy-5-hexenoate |
| CN112501235A (en) * | 2020-11-19 | 2021-03-16 | 华东师范大学 | Method for preparing breynolone by using monoalcohol ketone acetate |
| CN112501235B (en) * | 2020-11-19 | 2022-12-02 | 华东师范大学 | Method for preparing breynolone by using monoalcohol ketone acetate |
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Application publication date: 20200922 |