Method for efficiently preparing tauroursodeoxycholic acid through multiple cells

Abstract

The invention discloses a method for efficiently preparing tauroursodesoxycholic acid from multiple cells, and relates to the technical field of biological enzyme catalysis. The method solves the technical problems of low conversion rate, high cost, long reaction time, difficulty in industrial production and the like of an existing method for preparing the tauroursodesoxycholic acid. According to the method, a mixed enzyme solution is adopted as an enzyme system required in the reaction process to directly react to prepare the tauroursodeoxycholic acid, the mixed enzyme solution is enzyme-containing whole cells, the enzyme-containing whole cells are adopted, so that the enzyme activity is ensured, and the enzyme cost is greatly reduced; a micro-reaction concept is introduced, so that the reaction time is shortened to 30 minutes, and the time is saved; according to the preparation method, the preparation process is simplified, enzyme is recycled, the cost is saved, the preparation efficiency is also remarkably improved, and the content and the purity of a prepared TUDCA product are relatively high and reach 98% or above.

Description

technical field [0001] The invention relates to the technical field of biological enzyme catalysis, and more specifically relates to the technical field of a multi-cell efficient method for preparing tauroursodeoxycholic acid. Background technique [0002] Tauroursodeoxycholic acid (TUDCA), the chemical name is 3α, 7βdihydroxycholanoyl-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...

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Problems solved by technology

[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 generally complex, with low yield, high cost, poor environmental protection and safety. This is also the direct reason for the high price of TUDCA. The reason is that with the popularization of the domestic enzyme catalysis market in recent years, many manufacturers and research institutes have begun to try to produce TUDCA by enzymatic catalysis, but most of the current published literature and patents are only preliminary attempts of enzymatic catalysis, and there is still a long way to go before industrialized production. quite a distance

[0004] Invention patent CN107287272A discloses a method for preparing tauroursodeoxycholic acid, which respectively constructs expression vectors containing 7α-steroid dehydrogenase and 7β-steroid dehydrogenase or co-expression vectors of the two, in the culture medium Adding the substrate, transforming while fermenting, converting tauroursodeoxycholic acid into tauroursodeoxycholic acid, this method has low substrate concentration and low conversion rate, and the reaction intermediate taurine 7-ketolithiasis The acid content is high, the conversion cycle is long, and it is not easy to carry out industrial production; for the later purification method, the patent CN107287272A adopts the traditional extraction process, which cannot remove fat-soluble impurities, and uses reduced pressure concentration to remove organic solvents, which is not conducive to large-scale production

[0005] Invention patent CN102994604B discloses a two-step enzymatic method for preparing bound ursodeoxycholic acid, using recombinant enzymes 7α-HSDH and 7β-HSDH to catalyze bound CDCA (including TCDCA) in bile / powder in situ Generate bound state UDCA (comprising TUDCA), this method does not use the coenzyme regeneration system, adds a large amount of NADP+ and NADPH, the cost is very high, and the conversion rate is between 55%~60% in its embodiment, the conversion rate is too low, The post-extraction is difficult, the yield is low, and it does not have the conditions for large-scale production; in the specific method for later purification, the patent CN102994604B adopts multiple ultrasonic treatments, and at the same time uses a high-speed centrifuge and a rotary evaporator, which involves expensive equipment and facilities. And the operation is complicated, which is not conducive to large-scale production

[0006] Invention patent CN109402212A discloses a biotransformation method for preparing tauroursodeoxycholic acid and its application. In the first step, lactate dehydrogenase is used to catalyze α-keto acid to generate lactic acid, and at the same time, NADPH is converted into NADP+. In the second step, glucose is converted into sodium gluconate by glucose dehydrogenase, and NADP+ is converted 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, causing great pressure on the later sewage treatment. It is neither economical nor environmentally friendly; in the specific method for later purification, the patent CN109402212A uses a high-speed centrifuge and a rotary evaporator, which involves expensive equipment and complicated operations, which is not conducive to large-scale production

[0007] Invention patent CN112391419A discloses a biocatalytic preparation method of tauroursodeoxycholic acid, which uses organic solvents to participate in two-step enzymatic catalysis to generate TUDCA. Increase the production cost, and the reaction time in the embodiment exceeds 15h, and the production efficiency is low; in the specific method for later stage purification, the patent CN112391419A has adopted a centrifuge, vacuum concentrated organic solvent is the same as the general process, and used a 0.1 μm membrane filter According to the theory of industrialization, this membrane system has already involved in the field of ultrafiltration, and the use and maintenance costs are relatively high

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