A method for rapidly screening mutant strains of key intermediates in the synthesis of miglitol and bacterial strains

Abstract

The invention provides a method and bacterial strain for rapid quantitative detection of mutant strains of key intermediates of Miglitol. In the method, the mutagenized strains of wild Gluconobacter oxidans are subjected to fermentation and cultivation, and the fermented and cultivated mutant strains are obtained. Add the wet bacterial cells of the mutagenic strain to the substrate reaction solution. After the transformation reaction is complete at 15°C, centrifuge the reaction solution, measure the 6NSL content in the supernatant, and screen to obtain the key intermediate for the synthesis of miglitol according to the level of 6NSL content. highly active mutant strains. The detection method provided by the present invention has the characteristics of simplicity, quickness, high specificity, and good repeatability. The method can be further applied to the screening of high-activity Gluconobacter oxydans, effectively speeding up the breeding process, and transforming the mutant strains obtained through screening into synthetic rice. The catalytic ability of the glitol intermediate 6NSL has been significantly improved, and it is suitable for the industrialized large-scale production of miglitol.

Description

[0001] (1) Technical field [0002] The invention relates to a method for rapidly quantitatively detecting 6-deoxy-6-amino (N-hydroxyethyl)-α-L-sorbofuranosyl (6NSL), a key intermediate of miglitol. [0003] (2) Background technology [0004] Miglitol [1-(2-hydroxyethyl)-2-(hydroxymethyl)-3,4,5-piperidinetriol, miglitol], is a glucose structural analogue (such as figure 1 Shown), is a new type of α-glucosidase inhibitor hypoglycemic drug developed by Bayer (Bayer), Germany, which has high affinity for pancreatic amylase and α-glucosidase, and can inhibit disaccharides, polysaccharides and complexes. The hydrolysis of sugar delays the absorption of glucose and other monosaccharides, has obvious hypoglycemic effect, and has significantly lower toxic and side effects than sulfonamides and dimuscular drugs. It has become one of the important therapeutic drugs for the treatment of type II diabetes. [0005] US4246345, US4806650, US5401645, etc. disclose that the operational route f...

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Problems solved by technology

There are no relevant reports on the rapid quantitative detection of 6NSL in the existing patents and literature

In addition, although there are relevant reports on strains producing miglitol intermediates in China at present, such as CN101302549, CN105968042A, CN104693109A, etc., due to the small amount of bacteria in the fermentation unit of Gluconobacter oxydans, N-hydroxyethylglucosamine The activity of dehydrogenase is relatively weak, the catalytic process is slow, and the substrate concentration and conversion rate are low, which limits the production level of miglitol. It is urgent to establish an efficient screening method for high-activity strains, and miglitol The rapid quantitative detection method of the key intermediate 6NSL is a high-throughput screening method established by the technology platform, which can effectively speed up the breeding process of the high-activity catalytic synthesis of miglitol intermediate 6NSL strains, and further improve the production process of miglitol in my country lay the foundation

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