Biocatalytic preparation method of tauroursodeoxycholic acid

Abstract

The invention discloses a biocatalytic preparation method of tauroursodeoxycholic acid (TUDCA). The method comprises the following steps: step 1, performing dehydrogenation oxidation on TCDCA by usingTCDCA oxidase to obtain tauro 7-ketolithocholic acid (T-7-KLCA), and catalyzing acetone by using a ketone reductase to generate isopropanol to provide coenzyme regeneration of NADP+ / NAD+; and step 2,reducing T-7-KLCA into TUDCA by using a T-7-KLCA reductase, and catalyzing isopropanol by using isopropanol dehydrogenase to generate acetone to provide coenzyme regeneration of NADPH / NADH. Accordingto the double enzyme-linked reaction, after first-step reaction is completed, second-step reaction can be carried out through simple treatment, and intermediate purification is not needed; and the adding amount of NADP+ / NAD+ in the reaction is small, and acetone and isopropanol are substrates and products of each other in the two-step reaction and can be recycled, so that the production cost andthe environmental protection pressure are greatly reduced. An economic, efficient and environment-friendly large-scale industrial production new process is provided for generating TUDCA through doubleenzyme-linked reaction of taurochenodeoxycholic acid (TCDCA).

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a biocatalytic preparation method of tauroursodeoxycholic acid and a coenzyme regeneration system. Background technique [0002] Tauroursodeoxycholic acid (TUDCA), the chemical name is 3α, 7β dihydroxycholanoyl-N-taurine, which is formed by shrinking between the carboxyl group of ursodeoxycholic acid (UDCA) and the amino group of taurine. Conjugated bile acids. Discovered from bear bile in 1902, it is the main bile acid in bear bile and has antispasmodic, anticonvulsant, anti-inflammatory and gallstone-dissolving effects. Clinically, it is mainly used for the treatment of gallbladder cholesterol stones, primary sclerosing cholangitis, primary biliary cirrhosis and chronic hepatitis C virus. [0003] The current industrial production of TUDCA is generally a chemical process. Although the process has been continuously optimized and improved in recent years, the chemical process is gen...

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Problems solved by technology

Patent CN107287272A uses Escherichia coli expressing both 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes for liquid fermentation, and taurochenodeoxycholic acid Converted to tauroursodeoxycholic acid, although this method can obtain the final product, the time is too long, the patent states 1 to 3 days (preferably 2 to 3 days), and it is very difficult in the industrialized large-scale recombinant Escherichia coli fermentation process. It is easy to autolyze. Once the autolyzed substrate is recycled, it will be a problem. Therefore, this method is only a discussion of the enzyme-catalyzed process, and the economy and stability are difficult to meet the needs of industrial production.

Patent CN102994604B uses recombinant enzymes 7α-HSDH and 7β-HSDH to in situ catalyze the bound CDCA (including TCDCA) in bile / powder to generate bound UDCA (including TUDCA), but it does not use the coenzyme regeneration system, two-step enzyme catalysis A large amount of NADP+ and NADPH were added in the reaction, the cost of this alone has exceeded the value of the product, and the conversion rate in the examples is between 55% and 60%, the conversion rate is too low, the post-extraction is difficult, and the yield Low, no conditions for scale-up production

The coenzyme regeneration system in the patent CN109402212A two-step enzymatic reaction, the first step uses lactate dehydrogenase to catalyze α-keto acid to generate lactic acid, and at the same time converts NADPH into NADP+, and the second step uses glucose dehydrogenase to convert glucose into Sodium gluconate converts NADP+ into NADPH at the same time. These two steps generate a large amount of lactic acid and sodium gluconate, which cannot be recycled and are not easy to recycle. It will put a lot of pressure on the later sewage treatment, which is neither economical nor environmentally friendly.

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