Compound immobilized enzyme used for preparing statins and preparation method of compound immobilized enzyme

Abstract

The invention belongs to the technical field of medical industry, and mainly relates to a compound immobilized enzyme used for preparing statins, as well as a preparation method and applications of the compound immobilized enzyme. The compound immobilized enzyme provided by the invention is prepared through the manner of fixing active enzyme in an immobilized enzyme carrier through the fixing manner of adsorption, covalent binding, embedding, microencapsulation or crosslinking, wherein the active enzyme is composed of ketoreductase and / or halohydrin dehalogenase, and the mass percent of the ketoreductase in the active enzyme is 0%-100%. The immobilized enzyme product is applied to the process for producing the midbody, namely, ethyl(S)-4-chloro-3-hydroxybutanoate and / or ethyl(R)-4-cyano-3-hydroxybutyate of the products including atorvastatin, rosuvastatin, pitavastatin and the like. Compared with the liquid enzyme, the compound immobilized enzyme provided by the invention has the advantages that the production cost is reduced, the labor intensity is alleviated, the utilization efficiency of enzyme is improved, the discharge of waste water, waste gas and industrial residue is reduced, and the environment is protected.

Description

technical field

[0001] The invention relates to the fields of bioengineering and green pharmacy, in particular to a composite immobilized enzyme and its preparation method and application. Background technique

[0002] Statins are hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA) reductase by competitively inhibiting The metabolic pathway of intracellular valonate reduces intracellular cholesterol synthesis, thereby stimulating the increase in the number and activity of low-density lipoprotein (LDL) receptors on the surface of cell membranes (mainly liver cells) and clearing serum cholesterol increase, decrease in level. Statins can also inhibit the synthesis of apolipoprotein B-100 in the liver, thereby reducing the synthesis and secretion of triglyceride-rich AV and lipoproteins. The currently commercially available statins mainly include atorvastatin, rivastatin, and pitavasta...

Examples

Embodiment 1

[0037] Take a 250mL three-neck flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g ketoreductase, 40g epoxy-immobilized enzyme carrier (sepabeads® EC-EP113-M) in sequence, and the temperature is 25°C , rotate at 120 rpm, react for 24 hours, and collect the immobilized enzyme.

Embodiment 2

[0039] Take a 250mL three-neck flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g of halohydrin dehalogenase, 40g of epoxy-immobilized enzyme carrier (sepabeads® EC-EP113-M) in sequence, at a temperature of 30 °C, rotating speed 150rpm, react for 24 hours, and collect the immobilized enzyme.

Embodiment 3

[0041] Take a 250mL three-necked flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g ketoreductase, 40g activated amino-immobilized enzyme carrier (sepabeads® EC-HA / M) in sequence, at a temperature of 20 °C, rotating at 160 rpm, reacted for 24 hours, and collected the immobilized enzyme.

technical field

[0001] The invention relates to the fields of bioengineering and green pharmacy, in particular to a composite immobilized enzyme and its preparation method and application. Background technique

[0002] Statins are hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA) reductase by competitively inhibiting The metabolic pathway of intracellular valonate reduces intracellular cholesterol synthesis, thereby stimulating the increase in the number and activity of low-density lipoprotein (LDL) receptors on the surface of cell membranes (mainly liver cells) and clearing serum cholesterol increase, decrease in level. Statins can also inhibit the synthesis of apolipoprotein B-100 in the liver, thereby reducing the synthesis and secretion of triglyceride-rich AV and lipoproteins. The currently commercially available statins mainly include atorvastatin, rivastatin, and pitavasta...

Examples

Embodiment 1

[0037] Take a 250mL three-neck flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g ketoreductase, 40g epoxy-immobilized enzyme carrier (sepabeads® EC-EP113-M) in sequence, and the temperature is 25°C , rotate at 120 rpm, react for 24 hours, and collect the immobilized enzyme.

Embodiment 2

[0039] Take a 250mL three-neck flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g of halohydrin dehalogenase, 40g of epoxy-immobilized enzyme carrier (sepabeads® EC-EP113-M) in sequence, at a temperature of 30 °C, rotating speed 150rpm, react for 24 hours, and collect the immobilized enzyme.

Embodiment 3

[0041] Take a 250mL three-necked flask, add 150mL phosphate buffer solution (500mM, pH 8.0), and add 0.4g ketoreductase, 40g activated amino-immobilized enzyme carrier (sepabeads® EC-HA / M) in sequence, at a temperature of 20 °C, rotating at 160 rpm, reacted for 24 hours, and collected the immobilized enzyme.