阅读说明：本技术 一种低成本制备7-酮石胆酸的生物酶催化方法 (Bio-enzyme catalysis method for preparing 7-ketolithocholic acid at low cost ) 是由 傅荣昭 谷绪顶 周文俊 于 2021-08-27 设计创作，主要内容包括：本发明涉及一种低成本制备7-酮石胆酸的生物酶催化方法,该方法是将苏氨酸脱氨酶催化苏氨酸制备2-酮丁酸,然后2-酮丁酸作为辅底物,在7ɑ羟基类固醇脱氢酶、α-羟丁酸脱氢酶、鹅去氧胆酸以及辅酶NAD+的作用下,生成7-酮石胆酸,且酶催化的转化率能够达到99.5％以上,而且成本比现有工艺路线极具优势,有工业化应用的潜力。(The invention relates to a biological enzyme catalysis method for preparing 7-ketolithocholic acid at low cost, which is characterized in that threonine deaminase catalyzes threonine to prepare 2-ketobutyric acid, then the 2-ketobutyric acid is used as an auxiliary substrate, and 7-ketolithocholic acid is generated under the action of 7 alpha hydroxyl steroid dehydrogenase, alpha-hydroxybutyrate dehydrogenase, chenodeoxycholic acid and coenzyme NAD +, and the conversion rate of the enzyme catalysis can reach more than 99.5 percent, and the cost is superior to that of the existing process route and has the potential of industrial application.)

1. A biological enzyme catalysis method for preparing 7-ketolithocholic acid with low cost is characterized by comprising the following steps:

(1) taking chenodeoxycholic acid as a substrate, adding a biological catalyst, a cofactor and a coenzyme regeneration system into an aqueous phase reaction system at the temperature of 20-40 ℃ and the pH value of 6.0-8.0, and carrying out an oxidation reaction to generate a target product, namely 7-ketolithocholic acid;

(2) the biocatalyst is 7 alpha hydroxysteroid dehydrogenase; the coenzyme regeneration system comprises threonine, threonine deaminase and alpha-hydroxybutyrate dehydrogenase.

2. The method of claim 1, wherein: the chenodeoxycholic acid: the mass ratio of threonine to threonine deaminase to alpha-hydroxybutyrate dehydrogenase is 1: 2-3.2: 0.01-0.03: 0.05 to 0.2.

3. The method of claim 1, wherein: the cofactor in the step (1) is NAD⁺The cofactor is deoxygenated with the gooseThe mass ratio of the cholic acid to the raw materials is 0.0001-0.01: 1.

4. The method of claim 1, wherein: the enzymatic activity of the threonine deaminase in the step 1 is more than 3000U/g, the enzymatic activity of alpha-hydroxybutyrate dehydrogenase is more than 200U/g, and the enzymatic activity of 7 alpha hydroxysteroid dehydrogenase is more than 100U/g.

5. The method of claim 1, wherein: the threonine deaminase, the alpha-hydroxybutyrate dehydrogenase and the 7 alpha hydroxysteroid dehydrogenase are all expression products in escherichia coli.

Technical Field

The invention relates to a biological enzyme catalysis method for preparing 7-ketolithocholic acid at low cost, belonging to the field of biological enzyme catalysis.

Background

Ursodeoxycholic acid is mainly used for treating cholelithiasis, cholecystitis, cholangitis, and bile dyspepsia. Has good curative effect on bile reflux gastritis, alcoholic liver, biliary cirrhosis and hepatitis induced by drugs. Some developed western countries such as the United states are mainly used for preventing the formation of gallstones, and the diets of the countries take meat substances as main sources, have the characteristics of high energy and heat, density, grease enrichment and the like, have high probability of suffering from gallstones, and the ordinary people eat ursodeoxycholic acid to prevent the formation of the gallstones with the aim of prevention and the like. The prior preparation method of ursodeoxycholic acid mainly comprises a chemical synthesis method and a biotransformation method, wherein the most important intermediate for synthesizing the ursodeoxycholic acid is 7-ketolithocholic acid. The synthesis of 7-ketolithocholic acid has more routes, and in the chemical method, an oxidant is generally used for oxidizing CDCA to generate 7-ketolithocholic acid, and finally, product purification is required. The traditional method for chemically synthesizing 7-ketolithocholic acid has a series of problems of complex reaction process, low selectivity, harsh reaction conditions, large energy consumption, large pollution and the like, so that the industrial scale of UDCA is limited.

At present, a plurality of methods for preparing 7-ketolithocholic acid based on an enzyme catalysis technology exist, and the enzyme catalysis technology overcomes various defects of a traditional route. The enzymatic route for the preparation of 7-ketolithocholic acid requires coenzyme NAD⁺Regenerating, wherein substrates for coenzyme regeneration comprise sodium pyruvate, sodium malate and the like, the price of industrial sodium pyruvate is 80 yuan/kg, and the price of sodium malate is 30-40 yuan/kg. The substrate regenerated by the coenzyme is 2-ketobutyric acid, the threonine is prepared by the catalysis of threonine enzyme, the price of the threonine is 10 yuan/kg, and the cost of the preparation of the 2-ketobutyric acid by the catalysis of the threonine enzyme is not higher than 15 yuan/kg. Therefore, the threonine route selected by the coenzyme regeneration substrate has great industrial advantages.

Disclosure of Invention

Aiming at the technical problems, the invention provides a biological enzyme catalysis method for preparing 7-ketolithocholic acid at low cost, which can obviously reduce the cost and has great industrial advantages.

The invention achieves the purpose through the following steps:

a biological enzyme catalysis method for preparing 7-ketolithocholic acid with low cost is prepared by the following steps:

(1) taking chenodeoxycholic acid as a substrate, adding a biological catalyst, a cofactor and a coenzyme regeneration system into an aqueous phase reaction system at the temperature of 20-40 ℃ and the pH value of 6.0-8.0, and carrying out an oxidation reaction to generate a target product, namely 7-ketolithocholic acid;

(2) the biocatalyst is 7 alpha hydroxysteroid dehydrogenase; the coenzyme regeneration system comprises threonine, threonine deaminase and alpha-hydroxybutyrate dehydrogenase.

The chenodeoxycholic acid: the mass ratio of threonine to threonine deaminase to alpha-hydroxybutyrate dehydrogenase is 1: 2-3.2: 0.01-0.03: 0.05 to 0.2.

The cofactor in the step (1) is NAD⁺The mass ratio of the cofactor to the chenodeoxycholic acid is 0.0001-0.01: 1.

The enzymatic activity of the threonine deaminase in the step 1 is more than 3000U/g, the enzymatic activity of alpha-hydroxybutyrate dehydrogenase is more than 200U/g, and the enzymatic activity of 7 alpha hydroxysteroid dehydrogenase is more than 100U/g.

The threonine deaminase, the alpha-hydroxybutyrate dehydrogenase and the 7 alpha hydroxysteroid dehydrogenase are all expression products in escherichia coli.

The invention has the advantages of

(1) The conversion rate of the bio-enzyme catalysis can reach more than 99.5 percent.

(2) And the cost is superior to the existing process route, and the method has the potential of industrial application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail with reference to embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

10g of chenodeoxycholic acid and 4g of threonine are added into a reaction bottle, and then 50ml of water, 0.2g of threonine deaminase, 0.5g of alpha-hydroxybutyrate dehydrogenase, 2g of 7 alpha-hydroxysteroid dehydrogenase and NAD are added into the reaction bottle⁺0.01g, pH 7.5 at room temperature, reacting for 4-6h, and CDCA (chenodeoxycholic acid) finally remaining 0.5%.

After the reaction is finished, adjusting the acid to 4.0, heating to 70 ℃, then cooling to normal temperature, and filtering to obtain a filter cake. Adding 30ml of methanol into the filter cake, heating to 50 ℃, filtering to obtain filtrate, evaporating 15ml of methanol from the filtrate under reduced pressure, slowly dropwise adding 12ml of water, finally cooling to 5 ℃, filtering to obtain crystals, and drying to obtain 9.5g of the crystal.

Example 2

10g of chenodeoxycholic acid and 4g of threonine are added into a reaction bottle, and then 50ml of water, 0.2g of threonine deaminase, 0.5g of alpha-hydroxybutyrate dehydrogenase, 3g of 7 alpha-hydroxysteroid dehydrogenase and NAD are added into the reaction bottle⁺0.01g, ph 7.5 at room temperature, reacting for 4-6h, and CDCA (chenodeoxycholic acid) residue is 0.3%.

After the reaction is finished, adjusting the acid to 4.0, heating to 70 ℃, then cooling to normal temperature, and filtering to obtain a filter cake. Adding 30ml of methanol into the filter cake, heating to 50 ℃, filtering to obtain filtrate, evaporating 15ml of methanol from the filtrate under reduced pressure, slowly dropwise adding 12ml of water, finally cooling to 5 ℃, filtering to obtain crystals, and drying to obtain 9.6g of the filtrate.

Example 3

10g of chenodeoxycholic acid and 4g of threonine are added into a reaction bottle, 50ml of water, 0.2g of threonine deaminase, 0.5g of alpha-hydroxybutyrate dehydrogenase, 3g of 7 alpha hydroxysteroid dehydrogenase and 0.1g of NAD + are added into the reaction bottle, the pH is 7.5 at room temperature, the reaction lasts for 4-6h, and 0.1% of CDCA (chenodeoxycholic acid) is finally remained.

After the reaction is finished, adjusting the acid to 4.0, heating to 70 ℃, then cooling to normal temperature, and filtering to obtain a filter cake. Adding 30ml of methanol into the filter cake, heating to 50 ℃, filtering to obtain filtrate, evaporating 15ml of methanol from the filtrate under reduced pressure, slowly dropwise adding 12ml of water, finally cooling to 5 ℃, filtering to obtain crystals, and drying to obtain 9.5g of the crystal.