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

A technology for mutagenizing bacterial strains and wet bacteria, which is applied in the field of rapid screening of mutant strains and bacterial strains for key intermediates in the synthesis of miglitol, and can solve the problem of weak N-hydroxyethylglucosamine dehydrogenase activity and rapid quantification of 6NSL. Detect and limit the production level of miglitol, etc., to achieve the effect of accelerating the mutagenesis selection process, good genetic stability, and increasing the amount of screening

Active Publication Date: 2020-10-09
ZHEJIANG UNIV OF TECH +1
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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

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Rapid Determination of Miglitol Key Intermediate 6NSL Chromogenic Method

[0041] (1) Preparation of reagents:

[0042] Preparation of DNPH chromogenic solution (20mM): Weigh 39.6mg of AR grade DNPH (dinitrophenylhydrazone), add 10mL of 10M HCl aqueous solution to completely dissolve DNPH, dilute to 100mL with deionized water, and transfer the solution to the brown reagent In the bottle, keep away from light.

[0043] Preparation of NaOH aqueous solution (8M): Weigh 32 g of NaOH solid and dissolve it in 100 mL of deionized water.

[0044] Preparation of the substrate reaction solution: N-hydroxyethylglucamine 60g / L and magnesium sulfate heptahydrate 5g / L, concentrated hydrochloric acid to adjust the pH to 5.0, and the solvent is deionized water.

[0045] (2) Determination steps: Pipette 150 μL of the substrate reaction solution into a 96-well plate, add 25 μL, 20 mM DNPH chromogenic solution for thorough mixing, keep the temperature at 37 ° C for 15 min, add...

Embodiment 2

[0061] Example 2 Mutation Breeding and High-throughput Screening of Gluconobacter oxidans

[0062] (1) Strain activation: Connect Gluconobacter oxydans (Gluconobacter oxydans ZJB-605CCTCC No.M 208069, Patent Publication No. CN101591681) from a glycerol tube to the slant medium, and cultivate it in a constant temperature incubator at 28°C for 3 to 5 days; The mass final concentration composition of the matrix is ​​the same as in Example 1.

[0063] (2) Preparation of bacterial suspension and single spores: add 10mL 0.85% normal saline to the slant medium in step (1), scrape off the bacterial cells, pour the bacterial liquid into the Erlenmeyer flask with glass beads, shake After 10 minutes, the bacterium was dispersed, filtered into a sterilized empty Erlenmeyer flask with sterilized filter paper, and the filtered bacterium solution was diluted with sterile water, and the dilution factor was 10 -3 , which is the bacterial suspension;

[0064] (3) Ultraviolet mutagenesis: pipe...

Embodiment 3

[0077] Example 3 Comparison of the ability of Gluconobacter oxydans mutagen strain ZJB16009 and wild strain ZJB-605 to catalyze the synthesis of miglitol intermediate 6NSL

[0078] (1) Preparation of resting cells of Gluconobacter oxidans: Inoculate the slant medium (same as Example 1) with the starting strain Gluconobacter oxidans ZJB-605 and the mutagenized strain ZJB16009, cultivate at 28°C for 3 to 5 days, and wait for the slant culture CaCO 3 It becomes transparent and bright, and there is no bacteria in the naked eye. Pick the strains cultivated on the slant, inoculate the strains on the slant into a 250mL Erlenmeyer flask with 40mL seed culture solution (composition is the same as in Example 1), cultivate at 28°C and 235rpm for 48h, and obtain the seed solution, pH 4.0~7.0, OD 600 ≥8 reached the transition standard, and the microscopic examination showed short rod-shaped bacteria, dark coloring, and no miscellaneous bacteria. 2% inoculum by volume was transferred to t...

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/01C12N1/20C12P19/26C12R1/01
CPCC12N1/20C12N15/01C12P19/26C12N1/205C12R2001/01
Inventor 郑裕国柯霞汪宁宁余盼红胡忠策吴洋
Owner ZHEJIANG UNIV OF TECH
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