Halohydrin dehalogenase immobilization carrier and preparation method thereof

Abstract

The invention discloses a halohydrin dehalogenase immobilization carrier and a preparation method thereof, 100-120 parts by mass of chitosan, 30-40 parts by mass of cellulose ester, 10-15 parts by mass of carbon fiber, 1-2 parts by mass of nano metal and 20-25 parts by mass of hydrophilic polymer are compounded, and then a layer of cross-linking agent is coated on the surface to obtain the immobilization carrier for immobilization of halohydrin dehalogenase. The immobilized enzyme has the beneficial effects that the immobilized carrier can effectively enhance the stability of the immobilized enzyme, the immobilized enzyme has better chemical and thermal stability on the carrier, and the activity can be maintained under harsh conditions; the immobilized enzyme can be used for multiple times and is not easy to inactivate, so that the economic benefit is improved; the immobilized carrier allows operation in a wider pH and temperature range, and the catalytic reaction is optimized; loss of the enzyme in the reaction process is reduced through the immobilized carrier, the reaction efficiency can be effectively improved, separation and recovery of the enzyme become simple, and subsequent treatment and recovery are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field related to immobilized enzymes, in particular to a halohydrin dehalogenase immobilized carrier and a preparation method thereof. Background Art

[0002] Atorvastatin calcium (Atorvastaincalium), chemically named (3R,5R)-7-[2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)pyrrol-1-yl]-3,5-dihydroxyheptanoate calcium, is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Launched by Pfizer in the United States in 1997, it is a third-generation statin lipid-regulating drug and is widely used in clinical practice to prevent and treat hypercholesterolemia. This effect works by reducing the levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and apolipoprotein B (ApoB) in patients with dyslipidemia. In addition, atorvastatin calcium also has anti-inflammatory effects in atherosclerotic plaques. Due to its high efficiency and safety, it has always been one of the best-selling drugs for the treatment of hypercholesterolemia.

[0003] (R)-4-cyano-3-hydroxybutyric acid ethyl ester (Compound I) is a key intermediate compound in the synthesis of atorvastatin calcium. The preparation methods of (R)-4-cyano-3-hydroxybutyric acid ethyl ester include chemical method and biological method. At present, it is mainly produced by chemical method in China. The reaction conditions of chemical method are harsh, cost is high, purity is low, pollution is particularly serious, and the impact on the environment is very large. In comparison, the reaction conditions of biological method are mild, more practical and environmentally friendly. Halohydrin dehalogenase, also called halohydrin-hydrohalide lyase, catalyzes the conversion of aromatic or aliphatic ortho-halohydrins into epoxides and hydrogen halides through intramolecular affinity substitution mechanism, and is one of the key enzymes for microbial degradation of organic halogen compounds. Halohydrin dehalogenase forms hydrogen bonds between the conserved serine in the protein structure and the substrate hydroxyl oxygen atom, stabilizes the substrate binding, reduces the pKa value of tyrosine through arginine, and tyrosine acts as a nucleophile from the oxygen atom on the substrate to attack the carbon atom substituted by the ortho-halogen, thereby releasing the halogen atom and forming an epoxide. Halogen alcohol dehalogenase can not only catalyze the cleavage of carbon-halogen bonds to carry out dehalogenation reactions and synthesize epoxy compounds, but also can highly selectively catalyze and accept a series of non-natural nucleophiles other than halide ions, such as N3-, NO2-, CN-, etc. mediated epoxide ring-opening reactions to synthesize a series of optically pure chiral β-substituted alcohols such as azidated alcohols, cyano-substituted alcohols, nitro alcohol isothiocyanate-substituted alcohols. As an enzyme with multiple catalytic functions, halohydrin dehalogenase is widely used in important industrial fields such as pesticide chemical reagents, medicine and polymer chemistry, and is one of the most valuable industrial enzymes. Since free halohydrin dehalogenase cannot be reused, has poor stability, and is easily denatured and inactivated, the cost of halohydrin dehalogenase in the production process remains high. In addition, free halohydrin dehalogenase also has defects such as difficulty in subsequent separation from the product, which is not conducive to continuous production. These factors jointly restrict the large-scale use of halohydrin dehalogenase in industry. Summary of the invention

[0004] In view of the above shortcomings, the present invention adopts the following technical solutions: 1. A halohydrin dehalogenase immobilized carrier and a preparation method thereof, characterized in that: the immobilized carrier comprises the following components in parts by weight: Chitosan: 100-120 parts by mass; Cellulose ester: 30-40 parts by weight; Carbon fiber: 10-15 parts by mass; Nano metal: 1-2 parts by mass; Hydrophilic polymer: 20-25 parts by mass; The surface of the immobilized carrier is coated with a cross-linking agent; The preparation method of the immobilized carrier is as follows: S1. Dissolving chitosan in an acidic solvent in corresponding parts by mass to prepare a solution having a concentration of 2-3%; S2 corresponding parts by mass of cellulose ester is dissolved in a solvent to prepare a solution having a concentration of 1-2%; S3. Dissolve the corresponding mass parts of the hydrophilic polymer in water to the desired concentration, usually 1-2%; S4. The chitosan solution, the cellulose ester solution and the hydrophilic polymer solution are mixed and stirred evenly; S5. Add an appropriate amount of carbon fiber of corresponding mass parts to the mixed solution to ensure that the carbon fiber is evenly distributed in the solution; S6. Evenly dispersing the nano-metal particles in the mixed solution, using ultrasonic treatment or stirring to ensure that they are evenly dispersed and combined with the carrier matrix; S7. Spread the mixed solution evenly on a flat substrate (such as a glass sheet or a plastic film), use an oven at a suitable temperature to accelerate drying, gradually cool to room temperature, use a plastic scraper or soft tool to carefully peel the film from the substrate, and air-dry at room temperature for 3 to 5 hours; S8. Prepare a crosslinking agent 0.5wt% glutaraldehyde aqueous solution, soak the membrane in the solution for 20 to 30 minutes, then rinse with deionized water and heat to cure the crosslinking agent to allow it to fully react with the carrier to enhance structural stability; S9. The treated carrier is thoroughly dried to remove residual solvent and moisture, and then stored in a dry, cool, dark environment to maintain its stability and activity.

[0005] Furthermore, the cellulose ester is a mixture of one or more of cellulose acetate, cellulose propionate and cellulose carbonate.

[0006] Furthermore, the cross-linking agent is glutaraldehyde.

[0007] Furthermore, the nanometal is one or more of gold, silver, iron and copper.

[0008] Furthermore, the hydrophilic polymer is one or more of polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethyleneimine (PEI) and polyglycolic acid (PHA).

[0009] Furthermore, the oven drying temperature of step S7 is 40-60°C.

[0010] Furthermore, the step S6 is low-frequency 20-40 kHz ultrasound, and the ultrasound time is 5-10 minutes.

[0011] Furthermore, the acidic solution used in step S1 is acetic acid or 0.5-1 mol / L dilute hydrochloric acid.

[0012] Furthermore, the solvent used in step S2 is ethyl acetate.

[0013] Furthermore, the storage temperature of step S9 is 0-10°C.

[0014] The beneficial effects of the present invention are: 1. The immobilized carrier of the present invention can effectively enhance the stability of the enzyme after immobilization, and the immobilized enzyme on the carrier of the present invention has better chemical and thermal stability and can maintain activity under harsh conditions; 2. The enzyme immobilized by the present invention can be used multiple times without being easily inactivated, thereby improving economic benefits; 3. The immobilized carrier of the present invention allows operation within a wider pH and temperature range, thereby optimizing the catalytic reaction; 4. The present invention reduces the loss of the enzyme during the reaction process through the immobilized carrier, thereby effectively improving the reaction efficiency, making the separation and recovery of the enzyme simple, and facilitating subsequent treatment and recovery. DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] S1. Dissolve 100 parts by mass of chitosan in acetic acid to prepare a solution having a concentration of 2-3%; S2 corresponding to 30 parts by mass of cellulose ester dissolved in ethyl acetate solvent to prepare a solution having a concentration of 1-2%; S3. Dissolve 20 parts by mass of a hydrophilic polymer polyvinyl alcohol in water to the desired concentration, usually 1-2%; S4. The chitosan solution, the cellulose ester solution and the hydrophilic polymer solution are mixed and stirred evenly; S5. Add an appropriate amount of 10 parts of carbon fiber by mass to the mixed solution, stir and mix to ensure that the carbon fiber is evenly distributed in the solution; S6. Evenly disperse 1 part of nano iron powder in the mixed solution, and use 20-40kHz low-frequency ultrasonic treatment for 5-10 minutes to ensure that it is evenly dispersed and combined with the carrier matrix to obtain a mixed solution; S7. Spread the mixed solution evenly on a flat substrate (such as a glass sheet or a plastic film), use an oven at a suitable temperature to accelerate drying, gradually cool to room temperature, use a plastic scraper or soft tool to carefully peel the film from the substrate, and air-dry at room temperature for 3 to 5 hours; S8. Prepare a crosslinking agent 0.5wt% glutaraldehyde aqueous solution, soak the membrane in the solution for 20 to 30 minutes, then rinse with deionized water and heat to cure the crosslinking agent to allow it to fully react with the carrier to enhance structural stability; S9. The treated carrier is thoroughly dried to remove residual solvent and moisture, and then stored in a dry, cool, dark environment to maintain its stability and activity.

[0017] S1. Dissolve 120 parts by mass of chitosan in acetic acid to prepare a solution having a concentration of 2-3%; S2 corresponding to 40 parts by mass of cellulose ester dissolved in ethyl acetate solvent to prepare a solution having a concentration of 1-2%; S3. Dissolve 25 parts by mass of a hydrophilic polymer polyvinyl alcohol in water to the desired concentration, usually 1-2%; S4. The chitosan solution, the cellulose ester solution and the hydrophilic polymer solution are mixed and stirred evenly; S5. Add an appropriate amount of 15 parts by mass of carbon fiber to the mixed solution, stir and mix to ensure that the carbon fiber is evenly distributed in the solution; S6. Evenly disperse 2 parts of nano iron powder in the mixed solution, and use 20-40kHz low-frequency ultrasonic treatment for 5-10 minutes to ensure that it is evenly dispersed and combined with the carrier matrix to obtain a mixed solution; S7. Spread the mixed solution evenly on a flat substrate (such as a glass sheet or a plastic film), use an oven at a suitable temperature to accelerate drying, gradually cool to room temperature, use a plastic scraper or soft tool to carefully peel the film from the substrate, and air-dry at room temperature for 3 to 5 hours; S8. Prepare a crosslinking agent 0.5wt% glutaraldehyde aqueous solution, soak the membrane in the solution for 20 to 30 minutes, then rinse with deionized water and heat to cure the crosslinking agent to allow it to fully react with the carrier to enhance structural stability; S9. The treated carrier is thoroughly dried to remove residual solvent and moisture, and then stored in a dry, cool, dark environment to maintain its stability and activity.

[0018] After testing, it was found that the carrier obtained by the above implementation method has a high loading capacity, good catalytic ability, long storage time, and good stability. After being used for more than 5 times, the activity is still maintained at more than 90%.

[0019] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any marking in a claim should not be considered as limiting the claim to which it relates.

[0020] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A halohydrin dehalogenase immobilized carrier and a preparation method thereof, characterized in that: The immobilized carrier comprises the following components in parts by weight: Chitosan: 100-120 parts by mass; Cellulose ester: 30-40 parts by weight; Carbon fiber: 10-15 parts by mass; Nano metal: 1-2 parts by mass; Hydrophilic polymer: 20-25 parts by mass; The surface of the immobilized carrier is coated with a cross-linking agent; The preparation method of the immobilized carrier is as follows: S1. Dissolving chitosan in an acidic solvent in corresponding parts by mass to prepare a solution having a concentration of 2-3wt%; S2 corresponding parts by mass of cellulose ester dissolved in a solvent to prepare a solution having a concentration of 1-2wt%; S3. Dissolve the corresponding mass of the hydrophilic polymer in water to a concentration of 1-2wt%; S4. The chitosan solution, the cellulose ester solution and the hydrophilic polymer solution are mixed and stirred evenly; S5. Add an appropriate amount of carbon fiber of corresponding mass parts to the mixed solution to ensure that the carbon fiber is evenly distributed in the solution; S6. Evenly dispersing the nano-metal particles in the mixed solution, using ultrasonic treatment or stirring to ensure that they are evenly dispersed and combined with the carrier matrix; S7. Spread the mixed solution evenly on a flat substrate (such as a glass sheet or a plastic film), use an oven at a suitable temperature to accelerate drying, gradually cool to room temperature, use a plastic scraper or soft tool to carefully peel the film from the substrate, and air-dry at room temperature for 3 to 5 hours; S8. Prepare an aqueous solution of a crosslinker solution of glutaraldehyde, immerse the membrane in the solution for 20 to 30 minutes, then rinse with deionized water, heat and cure the crosslinker to allow it to fully react with the carrier to enhance structural stability; S9. The treated carrier is thoroughly dried to remove residual solvent and moisture, and then stored in a dry, cool, dark environment to maintain its stability and activity.

2. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The cellulose ester is a mixture of one or more of cellulose acetate, cellulose propionate and cellulose carbonate.

3. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The cross-linking agent is a 0.5 wt % aqueous solution of glutaraldehyde.

4. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The nano metal is one or more of gold, silver, iron and copper.

5. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The hydrophilic polymer is one or more of polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethyleneimine (PEI) and polyglycolic acid (PHA).

6. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The oven drying temperature of step S7 is 40-60°C.

7. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The step S6 is low-frequency 20-40 kHz ultrasound, and the ultrasound time is 5-10 minutes.

8. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The acidic solution used in step S1 is acetic acid or 0.5-1 mol / L dilute hydrochloric acid.

9. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The solvent used in step S2 is ethyl acetate.

10. The immobilized carrier of halohydrin dehalogenase and the preparation method thereof according to claim 1, characterized in that: The storage temperature in step S9 is 0-10°C.