A transformation strain and method for catalytically synthesizing miglitol intermediates

Abstract

The invention discloses a transformation strain and a method for catalyzing the synthesis of miglitol intermediates. The transformation strain is obtained from the starting strain ZJB16009 by transforming the recombinant expression plasmid pBBR-sldAB, and then screening, named Gluconobacter oxidans ( Gluconobacter oxydans), strain number pBBR‑sldAB, preservation number CCTCC No.M 2019033, its N‑hydroxyethylglucosamine dehydrogenase activity is greatly improved compared with the starting strain ZJB16009, and the space-time yield is about that of the starting strain ZJB16009 2 times, it shows that the Gluconobacter oxydans pBBR-sldAB of the present invention has higher potential application value in the catalytic synthesis of the key intermediate 6NSL of miglitol.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a transformation strain and a method for catalytically synthesizing an intermediate of miglitol. Background technique [0002] Miglitol [1-(2-hydroxyethyl)-2-(hydroxymethyl)-3,4,5-piperidine triol, miglitol] is a glucose structural analogue (as shown in formula I), It is a new type of α-glucosidase inhibitor hypoglycemic drug developed by Bayer, Germany. It has high affinity for pancreatic amylase and α-glucosidase, and can inhibit the hydrolysis of disaccharides, polysaccharides and complex sugars. It 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 treating type II diabetes. [0003] [0004] Patents US4246345, US4806650, US5401645, etc. have disclosed that using N-hydroxyethylg...

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Problems solved by technology

[0005] Researches at home and abroad, although there have been reports on strains for producing miglitol intermediates, especially related reports on the source strains of glucose oxidized, such as patent applications CN101302549A, CN105968042A, CN104693109A, etc. The activity of N-hydroxyethylglucosamine dehydrogenase in wild strains is relatively weak, and the tolerance to the substrate N-hydroxyethylglucosamine is poor

The catalytic process is slow, and the substrate concentration and conversion rate are low. As the concentration of N-hydroxyethylglucosamine used in CN101029321A is at 40g / L, the production level of miglitol is limited, and it is urgent to establish a high sorbitol desorbing method. Strains with hydrogenase activity

In addition, the cell immobilization of Gluconobacter oxydans or the repeated application strategy of resting cells (CN101270378A) can improve the yield of unit cells to a certain extent, but the resting cells are not sensitive to the substrate N-hydroxyethylglucosamine. The specific enzymatic activity of the selective dehydrogenation is not significantly improved, and the single batch of substrate feeding is 50g / L and below (Hu, ZC., Bu, JL., Wang, RY.et al.Appl Biochem Biotechnol (2018).https : / / doi.org / 10.1007 / s12010-018-2924-y)

Patent CN108441491A obtains high activity by establishing a rapid quantitative detection method for the key intermediate of miglitol 6-deoxy-6-amino(N-hydroxyethyl)-α-L-sorbfuranose (6NSL), combined with mutagenesis screening Catalytic synthesis of miglitol intermediate 6NSL strain ZJB16009, the substrate concentration increased to 60-80 / L, if the substrate concentration is further increased, the yield of product 6NSL will be significantly reduced

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