Transforming bacterium strain and method for catalytically synthesizing miglitol intermediate

A technology for miglitol and intermediates, which is applied in the field of transforming bacterial strains that catalyze the synthesis of miglitol intermediates, can solve the problem of poor tolerance of N-hydroxyethylglucosamine, no significant increase in specific enzyme activity, wild strain N - Problems such as weak activity of hydroxyethylglucosamine dehydrogenase

Active Publication Date: 2019-07-16
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 (CN1...

Method used

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  • Transforming bacterium strain and method for catalytically synthesizing miglitol intermediate
  • Transforming bacterium strain and method for catalytically synthesizing miglitol intermediate
  • Transforming bacterium strain and method for catalytically synthesizing miglitol intermediate

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Experimental program
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Embodiment 1

[0025] Construction of Gluconobacter oxydans recombinant strain Gluconobacter oxydans pBBR-sldAB (CCTCC No.M2019033).

[0026] (1) Molecular construction of recombinant expression plasmid pBBR-sldAB:

[0027] The coding sequence (GenBank number is EF493853.1) of the large and small subunits SldA (protein sequence accession number ABP65281.1) and SldB (protein sequence accession number ABP65282.1) of the sorbitol dehydrogenase localized to the cell membrane of Gluconobacter oxidans, The substitution of one or several nucleotides will cause the substitution of corresponding amino acid residues, thereby affecting the function of N-hydroxyethylglucosamine dehydrogenase. The coding sequence was inserted into the middle of SEQ ID No.1 (promoter sequence) and SEQ ID No.2 (terminator sequence), and inserted into the broad host expression vector pBBR1MCS-5 using restriction endonuclease sites XhoI and EcoRI to construct Obtain pBBR-sldAB recombinant expression plasmid.

[0028] (2) P...

Embodiment 2

[0048] Comparison of N-hydroxyethylglucosamine dehydrogenase activity between Gluconobacter oxydans pBBR-sldAB (CCTCC No.M 2019033) and the mutant strain ZJB16009 (CCTCC No.M201703).

[0049] (1) Preparation of resting cells of Gluconobacter oxydans: the starting strain Gluconobacter oxydans mutagenic strain ZJB16009 and the recombinant strain Gluconobacter oxydans pBBR-sldAB constructed by the present invention were respectively inoculated into 250 mL Erlenmeyer flasks containing 40 mL of seed culture solution Among them, the composition of the seed culture solution is D-sorbitol 50g / L, yeast extract powder 25g / L, KH 2 PO 4 5g / L, K 2 HPO 4 5g / L, the solvent is water, and the pH value is natural (referring to no need to adjust the pH). Cultivate at 28°C and 235rpm for 48 hours to obtain seed liquid, centrifuge at 10,000rpm for 10min, discard the supernatant, wash with water once, centrifuge and discard the supernatant, and precipitate into resting cells for subsequent tra...

Embodiment 3

[0061] The effect of different amino acids on the synthesis of sorbitol dehydrogenase by fermentation cultured Gluconobacter oxydans was investigated.

[0062] As can be seen from Example 2, although the N-hydroxyethylglucosamine dehydrogenase activity of the recombinant bacteria has been significantly improved, the content of the extracellular coenzyme PQQ is significantly lower than that of the starting strain (23.32vs.39.93mg / L) , suggesting that limited PQQ content may inhibit the improvement of N-hydroxyethylglucosamine dehydrogenase activity in cells. In order to verify this hypothesis, the corresponding dehydrogenase activity was determined by adding different concentrations of PQQ to the culture supernatant of the recombinant bacteria. From figure 1 It can be seen that with the increase of the exogenous PQQ content, the corresponding N-hydroxyethylglucosamine dehydrogenase activity is also significantly improved, confirming that the synthesis of PQQ in the recombinant...

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Abstract

The present invention discloses a transforming bacterium strain and a method for catalytically synthesizing a miglitol intermediate. The transforming bacterium strain is obtained by transforming a recombinant expression plasmid pBBR-sldAB into a starting bacterium strain ZJB16009 and then conducing screening, and named as gluconobacter oxydans, has a strain number of pBBR-sldAB and a preservationnumber of CCTCC No.M2019033, is significantly higher in N-hydroxyethylglucosamine dehydrogenase activity than the starting bacterium strain ZJB16009, and also has a space-time yield about 2 times of the starting bacterium strain ZJB16009. The gluconobacter oxydans pBBR-sldAB has relatively high potential application value in catalytic synthesis of a key intermediate 6NSL of the 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...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/01
CPCC12P19/26C12N1/205C12R2001/01
Inventor 郑裕国柯霞余盼红胡忠策吴洋陈亮
Owner ZHEJIANG UNIV OF TECH
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