CN111686809A - Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biopharmaceuticals, and specifically discloses a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and a preparation method and application thereof. In the present invention, carbonyl reductase and isopropanol dehydrogenase are co-embedded in a solid phase carrier obtained from polyvinyl alcohol and polyethylene glycol, and the obtained co-immobilized enzyme catalyst is catalyzed by carbonyl reductase and isopropanol dehydrogenation. The enzyme can achieve efficient and high stereoselective reduction of 3-carbonyl-5-hexenoate to generate ( R )-3-hydroxy-5-hexenoate in the cycle of coenzyme NADPH. The catalyst can be used to prepare ( R )-3-hydroxy-5-hexenoate by using 3-carbonyl-5-hexenoate as a substrate, and the co-immobilized catalyst prepared by the method of the present invention has high catalytic efficiency and stability It is good, reusable, simple in process, low in cost, and has excellent practical industrial application value.

Description

Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method thereof application

technical field

The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a co-immobilized enzyme catalyst, a preparation method thereof, and an application in the synthesis of ( R )-3-hydroxy-5-hexenoate.

Background technique

( R )-3-Hydroxy-5-hexenoate is an important chiral compound widely used in the synthesis of pharmaceutical and chemical intermediates including statins hypolipidemic drugs. Its structural formula is shown in Figure (I) below, in which R is C ₁ -C ₈ alkyl or cycloalkyl, mono- or polysubstituted aryl or aralkyl.

As the main way to synthesize compound (I) and its structural analogs, chemical methods have their shortcomings, such as extreme reaction conditions, including -20 ^o C to -50 ^o C low temperature (US patent US6355822) and High hydrogen pressure (European patent EP1176135), etc. These harsh reaction conditions severely limit the industrial prospects of chemical preparation of such compounds.

Our team reported the efficient and stereoselective (>99% ee ) reduction of compound (II) by carbonyl reductase to generate ( R )-3-hydroxy-5-hexenoate (I) (Chinese Patent Application CN107119081A, US patent US10526622). 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, the carbonyl reductase used for the catalytic reaction and the isopropanol dehydrogenase used for the regeneration of the coenzyme are both crude enzyme liquids, which make the post-reaction treatment complicated, and because the enzyme catalyst is difficult to recover and reuse, Increased industrial production costs.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst with high catalytic efficiency and good stability and a preparation method thereof and a method for synthesizing ( R )-3-hydroxy-5-hexenoic acid ester applications.

The preparation method of the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst provided by the invention comprises the following steps:

Step 1: prepare polyvinyl alcohol and polyethylene glycol into an aqueous solution of a certain concentration, heat to dissolve, and cool down to below 50 °C;

Step 2: Add a certain proportion of carbonyl reductase crude enzyme solution and isopropanol dehydrogenase crude enzyme solution (see our previous Chinese patent application CN107119081A for the preparation method of the crude enzyme solution) in the solution obtained in step 1, and mix evenly;

Step 3: After mixing, drop the solution onto the polyethylene film with a syringe, and then place it in a forced air oven at 35-40°C to dry for a certain period of time to obtain a co-immobilized catalyst for carbonyl reductase/isopropanol dehydrogenase , and stored at 4°C until use.

The amino acid sequence of carbonyl reductase is shown in SEQ ID NO.1, and the amino acid sequence of isopropanol dehydrogenase is shown in SEQ ID NO.2.

In step 1 of the present invention, preferably, the amount-to-mass ratio of the polyvinyl alcohol to polyethylene glycol is 5:3-5:1, more preferably 5:3-5:2.

In step 2 of the present invention, preferably, the initial concentrations of the crude carbonyl reductase enzyme solution and the crude isopropanol dehydrogenase enzyme solution are both 10%-30% (w/v), more preferably 15%-20% (w/v).

In step 2 of the present invention, preferably, the volume ratio of the crude carbonyl reductase enzyme solution, the crude isopropanol dehydrogenase 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 present invention, the drying time is 0.5-1 h.

In the present invention, carbonyl reductase and isopropanol dehydrogenase are co-embedded in a solid phase carrier obtained from polyvinyl alcohol and polyethylene glycol, and the obtained co-immobilized enzyme catalyst is catalyzed by carbonyl reductase and isopropanol dehydrogenation. Enzyme for the recycling of coenzyme NADPH to achieve efficient and highly stereoselective reduction of 3-carbonyl-5-hexenoate (II) to generate ( R )-3-hydroxy-5-hexenoate (I).

The co-immobilized enzyme catalyst prepared by the invention can be used for preparing ( R )-3-hydroxy-5-hexenoate (I) by using 3-carbonyl-5-hexenoate (II) as a substrate, specifically The steps are:

The co-immobilized enzyme catalyst, phosphate buffer, isopropanol, coenzyme NADP ⁺ and the substrate 3-carbonyl-5-hexenoate (II) were mixed, and the pH of the reaction solution was 6-9 after catalytic reaction. The reaction temperature is 15-35 ^o C to obtain ( R )-3-hydroxy-5-hexenoate (I);

Reaction formula of the present invention is:

In the formula, R is C ₁ -C ₈ alkyl or cycloalkyl, mono- or poly-substituted aryl or aralkyl.

As shown in the above reaction formula, in the reaction, the substrate 3-carbonyl-5-hexenoate (II) undergoes a reduction reaction under the catalysis of the co-immobilized enzyme catalyst to generate the product ( R )-3-hydroxy-5-hexene The acid ester (I), after the completion of the reaction, is separated and purified from the reaction solution to obtain the target product.

Preferably, in the initial reaction system, the mass percentage concentration of the substrate is 1%-30% (w/v), more preferably 10%-20%.

Preferably, in the initial reaction system, the percentage concentration of isopropanol is 5%-25% (v/v), more preferably 10%-20%.

Preferably, the amount of the co-immobilized enzyme catalyst is 20%-100% (w/w) of the mass of the substrate, more preferably 50%-100%.

Preferably, the amount of the coenzyme NADP ⁺ is 0.003%-0.01% (w/w) of the amount of the substrate.

Preferably, the reaction temperature is 25-30 ^℃ ;

Preferably, the pH of the reaction solution is 6.5-7.5.

After the above reaction is completed, the reaction solution is subjected to post-processing to obtain a finished product. The post-processing is as follows: filtration and separation of the co-immobilized enzyme catalyst, and the filtrate is extracted three times with ethyl acetate; the organic layers are combined and washed with water and saturated brine respectively, Dry with anhydrous sodium sulfate and concentrate to dryness under reduced pressure to obtain the finished product.

Compared with the prior art, the catalyst of the present invention has high catalytic efficiency, good stability, and can be reused. By comparing the co-immobilized catalyst and the free enzyme in terms of temperature stability, pH stability, and reusable times, the total The immobilized enzyme catalysts showed more excellent stability and catalytic efficiency. The invention has simple process, low cost and great industrial application value.

Description of drawings

Figure 1 shows the temperature stability curves of isopropanol dehydrogenase free enzyme and co-immobilized enzyme catalyst.

Figure 2 shows the temperature stability curves of carbonyl reductase free enzyme and co-immobilized enzyme catalysts.

Figure 3 is the pH stability curve of isopropanol dehydrogenase free enzyme and co-immobilized enzyme catalyst.

Figure 4 shows the pH stability curves of carbonyl reductase free enzyme and co-immobilized enzyme catalysts.

Figure 5 shows the storage stability curves of isopropanol dehydrogenase free enzyme, carbonyl reductase free enzyme and co-immobilized enzyme catalysts.

Figure 6 is a study on the reuse performance of the co-immobilized catalyst.

Detailed ways

The present invention will be described in further detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

Embodiment 1, the preparation of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst

Weigh 5 g of polyvinyl alcohol, 3 g of polyethylene glycol and 35 mL of water into a reaction flask, heat until the solution is clear, cool down to below 50 °C, add 10 mL of crude carbonyl reductase enzyme solution (15% w/v) and 5 mL of isopropyl alcohol. Propanol dehydrogenase crude enzyme solution (15% w/v), mix well. After mixing, the solution was added dropwise to the polyethylene film with a syringe, and then placed in a blast oven at 35-40 °C for 1 h to obtain the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which was stored in Reserve at 4°C.

Example 2, Thermal stability study of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst

Take carbonyl reductase free enzyme (crude enzyme solution), isopropanol dehydrogenase free enzyme (crude enzyme solution) and the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in Example 1, respectively, at 20 ℃、30℃、40℃、50℃、60℃、70℃、 80℃ 1 and 2 are shown.

Enzyme activity determination method of carbonyl reductase: take a certain amount of carbonyl reductase free enzyme or immobilized enzyme and add it to a phosphate buffer solution (100 mM, pH 7.5) containing substrate (10 mM) and NADPH (0.24 mM). ), shake for 1 min, centrifuge, take the supernatant to measure the change of absorbance at 340 nm, and calculate the enzyme activity per gram of enzyme.

Determination method of the enzyme activity of isopropanol dehydrogenase: take a certain amount of isopropanol dehydrogenase free enzyme or immobilized enzyme, add it to the solution containing isopropanol (2% v/v), NADP ⁺ (0.4 mM). In phosphate buffer solution (100 mM, pH 7.5), after shaking for 1 min, centrifuge, take the supernatant to measure the change of absorbance at 340 nm, and calculate the enzyme activity per gram of enzyme.

It can be seen from Figures 1 and 2 that for the enzyme activity of isopropanol dehydrogenase, the free enzyme loses its activity at temperatures above 50 °C, while the immobilized enzyme still retains 20% of its activity at 80 °C, and at a temperature of 40 °C to 80 °C. The temperature stability of the immobilized enzyme in the range is higher than that of the free enzyme. For carbonyl reductase, the temperature stability of the immobilized enzyme in the temperature range of 30-70 °C is higher than that of the free enzyme. The results show that the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is more stable than the free enzymes of isopropanol dehydrogenase and carbonyl reductase in high temperature environment, and can better maintain the catalytic activity.

Example 3, pH stability study of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst

Take carbonyl reductase free enzyme, isopropanol dehydrogenase free enzyme and carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in Example 1, add to pH 4, 5, 6, 7, 8 In the buffer solution, incubate in 30℃ water bath for 5min, then measure the enzyme activity respectively, take the highest activity as 100%, measure in parallel three times, and make the corresponding pH stability curve, as shown in Figures 3 and 4.

It can be seen from Figures 3 and 4 that for isopropanol dehydrogenase, both the immobilized enzyme and the free enzyme are inactive at pH=4, while in the case of pH=5-8, the pH stability of the immobilized enzyme Both were improved compared with free enzyme. For carbonyl reductase, the pH stability of the immobilized enzyme was lower than that of the free enzyme under acidic conditions; however, the pH stability of the immobilized enzyme was higher than that of the free enzyme under alkaline conditions. The results show that under alkaline conditions, the co-immobilized catalyst of carbonyl reductase/isopropanol dehydrogenase is more stable than the free enzymes of isopropanol dehydrogenase and carbonyl reductase, and can better maintain the catalytic activity.

Example 4, Storage stability study of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst

Take carbonyl reductase free enzyme, isopropanol dehydrogenase free enzyme and carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in Example 1, and place it at 20 ° C for 12 days. The initial activity is 100%, the enzymatic activities of 4 days and 12 days were measured respectively, and curve 5 was obtained.

It can be seen from Figure 5 that after 12 days of storage, the stability of the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is higher than that of the free enzyme. Among them, the enzymatic activity of carbonyl reductase of the co-immobilized catalyst remained basically unchanged, and the enzymatic activity of isopropanol dehydrogenase remained above 85%. The activities of carbonyl reductase free enzyme and isopropanol dehydrogenase free enzyme both dropped below 80%, and the activity of isopropanol dehydrogenase free enzyme was only 60%. The results show that the storage stability of the immobilized enzyme at room temperature is better than that of the free enzyme, and it can be stored for a long time.

Example 5. Research on the reusability of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst

Take the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (100 mg) prepared in Example 1, add the substrate tert-butyl 3-carbonyl-5-hexenoate (100 mg), isopropanol (0.1 mL), NADP ⁺ (0.01 mg), phosphate buffer (50 mM, pH=7.0) (0.4 mL), reacted in a constant temperature shaking shaker for 10 h (30 °C, 200 rpm). The conversion rate of the substrate was measured, and then the co-immobilized enzyme catalyst was recovered by filtration and washed. The co-immobilized enzyme catalyst was reused 18 times in this way, the conversion rate was measured, and the reuse performance chart was drawn (Fig. 6).

It can be seen from Figure 6 that the substrate conversion rate of the immobilized enzyme is basically stable (>98%) after 10 repeated use; after 18 repeated use, the substrate conversion rate still remains above 85%. The results show that the enzyme embedded in the immobilized carrier is relatively stable, not easy to fall off, and the enzyme activity remains stable, which can be reused.

Example 6, Carbonyl reductase/isopropanol dehydrogenase co-supported catalyst catalyzed asymmetric reduction to generate ( R )-3-hydroxy-5-hexenoic acid tert-butyl ester (gram scale)

Take the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (1 g) prepared in Example 1, and add the substrate tert-butyl 3-carbonyl-5-hexenoate (1 g) and isopropanol (1 mL) , NADP ⁺ (0.1 mg), phosphate buffer (50 mM, pH=7.0) (4 mL), react in a constant temperature shaking shaker for 10 h (30 °C, 200 rpm), after the reaction, filter and recover the co-immobilized enzyme catalyst . The filtrate was extracted three times with ethyl acetate, the organic layers were combined, washed with water and saturated brine respectively, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 0.91 g of the finished product (yield 90%, ee value 99.7%). ¹ H NMR (CDCl ₃ , 400 MHz): δ/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, Carbonyl reductase/isopropanol dehydrogenase co-supported catalyst catalyzed asymmetric reduction to generate ( R )-3-hydroxy-5-hexenoic acid methyl ester (gram scale)

Take the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst (1 g) prepared in Example 1, add the substrate 3-carbonyl-5-hexenoic acid methyl ester (1 g), isopropanol (0.8 mL), NADP ⁺ (0.07 mg), phosphate buffer (50 mM, pH=7.0) (4 mL) were reacted in a constant temperature shaking shaker for 12 h (30 °C, 200 rpm). After the reaction, the co-immobilized enzyme catalyst was recovered by filtration. The filtrate was extracted three times with ethyl acetate, the organic layers were combined, washed with water and saturated brine respectively, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 0.90 g of the finished product (yield 89%, ee value 99.8%). ¹ H NMR (CDCl ₃ , 400 MHz): δ/ppm 5.81 (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 is only the preferred embodiment of the present invention, only to illustrate the technical concept and characteristics of the present invention, and its purpose is to enable those who are familiar with the technology to understand the content of the present invention and implement it accordingly, and it should not be limited by this. protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made according to the spirit of the present invention shall be included within the protection scope of the present invention.

sequence listing

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<120> Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and its preparation method and application

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Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

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Lys Val Val Ile Thr Gly Arg Arg Ala Asp Val Gly Glu Arg Ala Ala

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Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Ile Gln His Asp Ala

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Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Asp Val Val

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Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Phe Gly Met Val Gly Asp Pro Thr Val Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys

180 185 190

Thr Pro Met Leu Asp Asp Val Glu Gly Ala Glu Glu Met Trp Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Val Cys Val Tyr Leu Ala Ser Gly Glu Ser Lys Phe Ala Thr Gly

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Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

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Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

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Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

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Claims (10)

1. a preparation method of carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, is characterized in that, concrete steps are: Step 1: prepare an aqueous solution of polyvinyl alcohol and polyethylene glycol, heat to dissolve, and cool down to below 50°C; Step 2: Add the crude enzyme solution of carbonyl reductase and the crude enzyme solution of isopropanol dehydrogenase to the solution obtained in step 1, and mix them evenly; Step 3: After mixing, drop the solution onto the polyethylene film with a syringe, and then place it in a forced air oven at 35-40°C to dry to obtain a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which is then stored. spare. 2. preparation method according to claim 1 is characterized in that, the consumption mass ratio of polyvinyl alcohol described in step 1 and polyethylene glycol is 5:3-5:1. 3. preparation method according to claim 1 is characterized in that, the initial concentration of used carbonyl reductase crude enzyme liquid and isopropanol dehydrogenase crude enzyme liquid in step 2 is 10%-30% (w/v ). 4. preparation method according to claim 1 is characterized in that, the volume ratio of carbonyl reductase crude enzyme liquid, isopropanol dehydrogenase crude enzyme liquid, polyvinyl alcohol/polyethylene glycol aqueous solution used in step 2 is 2:1:5-2:1:10. 5. The preparation method according to claim 1, wherein the drying time in step 3 is 0.5-1 h. 6. A carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst obtained by the preparation method of one of claims 1-5. 7. The application of the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst according to claim 6 in the preparation of ( R )-3-hydroxy-5-hexenoate. 8. application according to claim 7, is characterized in that, concrete steps are: The co-immobilized enzyme catalyst, phosphate buffer, isopropanol, coenzyme NADP ⁺ and the substrate 3-carbonyl-5-hexenoate (II) were mixed, and the pH of the reaction solution was 6-9 after catalytic reaction. The reaction temperature is 15-35 ^o C to obtain ( R )-3-hydroxy-5-hexenoate (I); The reaction formula is:

In the formula, R is C ₁ -C ₈ alkyl or cycloalkyl, mono- or poly-substituted aryl or aralkyl. 9. application according to claim 8, is characterized in that, in initial reaction system, the mass percentage concentration of substrate is 1%-30% (w/v); The percentage concentration of isopropanol is 5% %-25% (v/v). 10. The application according to claim 8, wherein the consumption of the co-immobilized catalyst is 20%-100% (w/w) of the mass of the substrate; the consumption of the coenzyme NADP ⁺ is the consumption of the substrate 0.003%-0.01% (w/w).

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