Halohydrin dehalogenase curing method

Abstract

The invention relates to a halohydrin dehalogenase curing method, which comprises the following steps: dissolving halohydrin dehalogenase in a polyvinylpyrrolidone aqueous solution, and adding urea and formaldehyde to carry out curing reaction to obtain immobilized halohydrin dehalogenase. According to the method, the immobilized enzyme mass ratio of the halohydrin dehalogenase can be increased, the enzyme titer per unit mass can be increased, the enzymatic reaction yield can be increased, the immobilized product of the halohydrin dehalogenase is stable, the enzymatic reaction process is high in controllability, and the yield and purity of ATS-5 enzyme reaction can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation extraction, and particularly relates to a method for solidifying halohydrin dehalogenase. Background Art

[0002] Atorvastatin, a novel lipid-lowering statin drug marketed under the trade name Lipitor, is a novel 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Numerous synthetic routes exist for atorvastatin, including the Paal-Knorr method, asymmetric carbonyl reduction, racemic phenylethylamine resolution, and selective aldol condensation. Compared to other methods, the Paal-Knorr method offers simplicity, mild reaction conditions, low cost, controllable quality, and high yield, making it the primary route for synthesizing atorvastatin APIs. Within the Paal-Knorr synthesis route, (R)-4-cyano-3-hydroxybutyric acid ethyl ester (ATS-5) is an important intermediate.

[0003] The enzymatic preparation of ATS-5 is green and environmentally friendly, with reliable quality, and has become the development direction of the industry. Commonly used enzymes include halohydrin dehalogenase, lipase, nitrilase and carbonyl reductase. Among them, the optical purity of ATS-5 obtained by lipase catalysis is high, but the theoretical yield is about 50%, which limits its industrial application. Nitrilase can catalyze the symmetrical generation of ATS-5 from 3-hydroxyglutaronitrile. However, when the substrate concentration is high, it will produce substrate inhibition on the enzyme, thus limiting its large-scale production. Carbonyl reductase is in the coenzyme NAD. In the presence of hydrogen, 2-methyl-4-chloro-3-oxybutanoic acid can be catalyzed to produce ATS-5. However, the expensive addition of the coenzyme NADH increases production costs, limiting its industrial application prospects. Compared with these three enzymes, halohydrin dehalogenase (HHDH) offers certain advantages. Without the need for any coenzyme, HHDH catalyzes the removal of the halogen element from the substrate (S)-ethyl 4-chloro-3-hydroxybutyrate (ATS-4), forming an epoxide. The epoxide is then attacked by a cyanide group and converted to ATS-5. This reaction occurs in aqueous solution. Since HHDH is difficult to completely remove from the reaction system after the reaction, subsequent purification processes are difficult, reducing the yield and optical purity of ATS-5. Therefore, to simplify the separation and purification process and simultaneously recover HHDH, research on immobilized HHDH is needed.

[0004]

[0005] Invention patent CN105316369A, a sodium alginate-immobilized halohydrin dehalogenase catalyzes the synthesis of (R)-4-cyano-3-hydroxybutyric acid ethyl ester (ATS-5). Sodium alginate is first mixed with HHDH, cross-linked with glutaraldehyde, and then embedded with calcium chloride. After immobilization for 1 hour, the immobilization rate of HHDH is above 46%. The immobilized enzyme is then reacted with the substrate (S)-4-chloro-3-hydroxybutyric acid ethyl ester (ATS-4). After 4 hours of reaction, the ATS-5 generation rate is above 70%. After recovering the HHDH, the catalytic reaction is carried out for the second time, and the ATS-5 generation rate is above 35%.

[0006] Invention patent CN105925558A, a composite immobilized enzyme for preparing statins and its preparation method, is prepared by immobilizing an active enzyme in an immobilized enzyme carrier in a covalently fixed manner, the immobilized enzyme carrier is selected from an epoxy immobilized enzyme carrier, wherein the active enzyme is composed of a ketone reductase and a halohydrin dehalogenase, the ketone reductase accounts for 20% to 80% of the mass percentage of the active enzyme, and the halohydrin dehalogenase accounts for 0.01% to 5% of the mass percentage of the immobilized enzyme carrier; the preparation method of the composite immobilized enzyme is: adding the active enzyme and the immobilized enzyme carrier to a buffer solution respectively, the buffer solution being selected from a phosphate buffer solution, a Tris-HCl buffer solution or a triethanolamine buffer solution.

[0007] In the above halohydrin dehalogenase immobilization process, the problems mainly include the following:

[0008] 1 Invention patent CN105316369A uses sodium alginate as a fixative and glutaraldehyde as an extractant. When the pH of the glutaraldehyde solution reaches 6, glutaraldehyde itself tends to polymerize. As the pH rises, this polymerization is extremely rapid, and precipitation will appear in the solution, affecting the solidification effect of sodium alginate.

[0009] 2 Invention patent CN105925558B, a composite immobilized enzyme is prepared, and the mass percentage of halohydrin dehalogenase in the immobilized enzyme carrier is 0.01% to 5%. The enzyme content is low, which significantly affects the catalytic reaction of ATS-4 to produce ATS-5. Summary of the Invention

[0010] The present invention provides a method for immobilizing a halohydrin dehalogenase. By carrying out a halohydrin dehalogenase immobilization reaction, the halohydrin dehalogenase can maintain its catalytic properties while being reusable, and can be easily separated from a product in a subsequent process, thereby having good industrial application prospects.

[0011] The technical solutions adopted to achieve the above objectives are:

[0012] The first object of the present invention is to provide a method for immobilizing a halohydrin dehalogenase, comprising the steps of dissolving the halohydrin dehalogenase in a polyvinylpyrrolidone aqueous solution, adding urea and formaldehyde to carry out a curing reaction, and obtaining an immobilized halohydrin dehalogenase.

[0013] Wherein, the curing method is characterized in that the pH value of the curing reaction is 8.2 to 8.5.

[0014] Wherein, the curing reaction temperature is 65-80°C.

[0015] Wherein, the curing reaction time is 90 to 120 minutes.

[0016] Wherein, the concentration of the polyvinyl pyrrolidone aqueous solution is 7-9% (W / W).

[0017] Wherein, the formaldehyde concentration is 37% (W / W).

[0018] Wherein, the mass ratio of the added urea to formaldehyde is 1:1.6-1.8.

[0019] The immobilization method of the halohydrin dehalogenase further comprises the step of adding acetone to the immobilized halohydrin dehalogenase for recrystallization and purification.

[0020] The recrystallization purification step refers to adding 3 to 5 times the volume of acetone to the immobilized halohydrin dehalogenase reaction solution, precipitating the immobilized halohydrin dehalogenase, filtering, and vacuum drying to obtain the immobilized halohydrin dehalogenase product.

[0021] Wherein, the pH value at which the curing reaction is terminated is 3.0 to 3.2.

[0022] Another object of the present invention is to provide a method for preparing (R)-4-cyano-3-hydroxybutyrate, using the immobilized halohydrin dehalogenase prepared by the above method as a catalyst, (S)-4-chloro-3-hydroxybutyric acid ethyl ester as a substrate, and a cyanide salt to carry out a catalytic reaction to obtain (R)-4-cyano-3-hydroxybutyrate.

[0023] The technical solution of the present invention has at least the following beneficial technical effects:

[0024] 1. This patent uses polyvinyl pyrrolidone with urea and formaldehyde for curing reaction, which can increase the mass ratio of immobilized halohydrin dehalogenase, increase the enzyme titer per unit mass, and improve the yield of enzymatic reaction;

[0025] 2. The polymerized solidified product of the halohydrin dehalogenase is stable, the enzymatic reaction process is highly controllable, it is easy to separate from the product, and continuous production can be achieved, effectively improving the reaction yield and purity of the ATS-5 enzyme. The purity of the reaction product reaches more than 96%, and the reaction yield reaches more than 97%. DETAILED DESCRIPTION

[0026] The present invention is described below with reference to examples. It should be understood that the examples are for the purpose of illustrating the present invention rather than limiting the present invention. The scope and core content of the present invention are determined by reference to the claims.

[0027] Preparation of 15% (W / W) polyvinyl pyrrolidone aqueous solution: Take 600g of purified water, heat it to about 40°C, add 150g of polyvinyl pyrrolidone while stirring, stir until dissolved, cool to room temperature, add water to 1000g, mix well, and obtain.

[0028] Take 60g of halohydrin dehalogenase (5 million u / g), divide it into 7 parts, and perform the following experiments respectively:

[0029] Example 1

[0030] Take 7g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 7% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.2, control the temperature at 65°C, add 4g of urea, add 17.3g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛 =1:1.6, stirring and reflux for 90 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.0 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 3 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 32 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0031] In a reaction flask, 300 g of purified water was added. Under stirring, 50 g of substrate ATS-4 and 2 g of sodium cyanide were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.2. When the temperature was raised to 50°C, 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 98% and a purity of 98%.

[0032] Example 2

[0033] Take 8.0g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 8% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.5, control the temperature at 75°C, add 4g of urea, add 18.4g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛=1:1.7, stirring and reflux for 100 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.2 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 4 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 34 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0034] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.4. When the temperature was raised to 50°C, 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 96% and a purity of 97%.

[0035] Example 3

[0036] Take 9.0g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 9% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.3, control the temperature at 78°C, add 4g of urea, add 19.4g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛 =1:1.8, stirring and reflux for 120 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.2 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 5 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 35 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0037] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.3. When the temperature was raised to 50°C, 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 98% and a purity of 97%.

[0038] Example 4

[0039] Take 8.5g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 8.5% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.3, control the temperature at 80°C, add 4g of urea, add 18g of 37% (w / w) formaldehyde, and add the amount according to W尿素 :W 甲醛 =1:1.67, stirring and reflux for 100 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.2 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 4 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 34 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0040] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.3. When the temperature was raised to 50°C, 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 98% and a purity of 99%.

[0041] Example 5

[0042] Take 8.0g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 8% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.3, control the temperature at 80°C, add 4g of urea, add 18.4g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛 =1:1.7, stirring and reflux for 110 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.1 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 3 to 5 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 34 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0043] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.5. When the temperature was raised to 50°C, 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 97% and a purity of 98%.

[0044] Comparative Example 1

[0045] In a reaction flask, 300 g of purified water was added, and under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.3. When the temperature was raised to 50°C, 5 g of free halohydrin dehalogenase was added, and the reaction was carried out for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.1. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 92.6% and a purity of 78.2%.

[0046] Comparative Example 2

[0047] Take 8.0g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 8.0% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.2, control the temperature at 70°C, add 4g of urea, add 14g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛 =1:1.5, stirring and reflux for 100 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.0 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 4 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 22 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0048] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.3. When the temperature was raised to 50°C, more than 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 67% and a purity of 72%.

[0049] Comparative Example 3

[0050] Take 8.0g of halohydrin dehalogenase, add 60g of 15% (w / w) polyvinyl pyrrolidone aqueous solution under stirring, stir until dissolved, add 15% (w / w) polyvinyl pyrrolidone aqueous solution to 100g, mix well, prepare 8.0% (w / w) halohydrin dehalogenase miscible solution, add 10% (w / w) liquid caustic soda to adjust pH8.5, control the temperature at 80°C, add 4g of urea, add 20.5g of 37% (w / w) formaldehyde, and add the amount according to W 尿素 :W 甲醛 =1:1.9, stirring and reflux for 100 minutes, adding 3 mol / L hydrochloric acid to adjust the pH to 3.2 to terminate the polymerization reaction to obtain a halohydrin dehalogenase immobilized reaction solution, adding 4 times the volume of acetone, the halohydrin dehalogenase immobilized product precipitated, filtered, and vacuum dried to obtain 25 g of polyvinyl pyrrolidone immobilized halohydrin dehalogenase.

[0051] In a reaction flask, 300 g of purified water was added. Under stirring, 2 g of sodium cyanide and 50 g of substrate ATS-4 were added in sequence. 10% (w / w) liquid caustic soda was added to adjust the pH to 8.5. When the temperature was raised to 50°C, more than 5 g of polyvinylpyrrolidone-immobilized halohydrin dehalogenase was added. The reaction was allowed to react for 4 h. 10% (w / w) liquid caustic soda was added to adjust the pH to 7. The mixture was extracted with ethyl acetate and distilled to obtain ATS-5 with a yield of 71% and a purity of 69%.

[0052] The above comparative cases were not carried out under the immobilized halohydrin dehalogenase reaction conditions specified in the present invention. The obtained immobilized halohydrin dehalogenase product had low quality and low halohydrin dehalogenase content, which affected the yield and purity of ATS-5 produced by the enzymatic reaction of ATS-4. The specific situation is shown in the table below:

[0053] Effect comparison:

[0054]

Claims

1. A method for immobilizing a halohydrin dehalogenase, comprising the steps of: dissolving the halohydrin dehalogenase in a polyvinylpyrrolidone aqueous solution, adding urea and formaldehyde to carry out a curing reaction, and obtaining an immobilized halohydrin dehalogenase.

2. The curing method according to claim 1, characterized in that The pH value of the curing reaction is 8.2-8.

5.

3. The curing method according to claim 1, characterized in that The curing reaction temperature is 65-80°C.

4. The curing method according to claim 1, characterized in that The curing reaction time is 90 to 120 minutes.

5. The curing method according to claim 1, characterized in that The concentration of the polyvinyl pyrrolidone aqueous solution is 7-9% (W / W).

6. The curing method according to claim 1, characterized in that The formaldehyde concentration was 37% (W / W).

7. The curing method according to claim 1, characterized in that The mass ratio of the added urea to formaldehyde is 1:1.6-1.

8.

8. The curing method according to claim 1, characterized in that The method also includes a purification step of adding acetone to the immobilized halohydrin dehalogenase for recrystallization. The recrystallization purification step refers to adding 3 to 5 times the volume of acetone to the immobilized halohydrin dehalogenase reaction solution, precipitating the immobilized halohydrin dehalogenase, filtering, and vacuum drying to obtain the immobilized halohydrin dehalogenase product.

9. The curing method according to claim 1, characterized in that The pH value at which the curing reaction is terminated is 3.0 to 3.

2.

10. A method for preparing (R)-4-cyano-3-hydroxybutyrate, characterized in that: The immobilized halohydrin dehalogenase prepared by the method of claim 1 is used as a catalyst, (S)-4-chloro-3-hydroxybutyric acid ethyl ester is used as a substrate, and a cyanide salt is used to carry out a catalytic reaction to obtain (R)-4-cyano-3-hydroxybutyrate.

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