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Fermentation method for high-content 12[alpha]-hydroxysteroid dehydrogenase fermentation liquor

A technology of hydroxysteroid and fermentation method, which is applied in the field of fermentation of fermented liquid with high content of 12α-hydroxysteroid dehydrogenase, can solve the problem of low activity and achieve the effect of high activity, high enzyme activity and reasonable nutrition mix

Inactive Publication Date: 2020-02-07
四川澄华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the defect that the above-mentioned existing 12α-hydroxysteroid dehydrogenase enzyme activity is only 50U / mL and the activity is low, the present invention provides a fermentation method of a high-content 12α-hydroxysteroid dehydrogenase fermentation broth

Method used

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Experimental program
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Effect test

Embodiment 1

[0029]A method for fermenting a high-content 12α-hydroxysteroid dehydrogenase fermented liquid, taking spare 12α-hydroxysteroid dehydrogenase expression gene recombinant E. Bacteria fermentation medium was cultivated, and induction medium was added after 2 hours for a total of 8 hours; the recombinant Escherichia coli expressing the 12α-hydroxysteroid dehydrogenase gene was a secondary fermentation strain, stored in glycerol tubes, and The fraction is 1-5% of the inoculum size, which is inserted into an Erlenmeyer shaker flask containing the LB liquid sterile medium, and placed in a shaker at 37°C, 40RH%, and 240rpm for 12 hours for later use; the new sterile fermentation medium, Percentage by weight, including corn flour 0.1%, corn steep liquor 2%, nitrate 0.05%, ammonium salt 0.05%, ammonia water 0.1%, phosphate 0.1%, sodium chloride 0.2%, magnesium sulfate 0.02%, zinc sulfate 0.02%, 0.05% defoamer, the balance is water; the induction medium, by weight percentage, includes 0...

Embodiment 2

[0031] A method for fermenting a high-content 12α-hydroxysteroid dehydrogenase fermented liquid, taking spare 12α-hydroxysteroid dehydrogenase expression gene recombinant E. Bacteria fermentation medium was cultivated, and induction medium was added after 2 hours for a total of 8 hours; the recombinant Escherichia coli expressing the 12α-hydroxysteroid dehydrogenase gene was a secondary fermentation strain, stored in glycerol tubes, and The fraction is 1-5% of the inoculum size, which is inserted into an Erlenmeyer shaker flask containing the LB liquid sterile medium, and placed in a shaker at 37°C, 45RH%, and 240rpm for 12 hours for later use; the new sterile fermentation medium, Percentage by weight, including corn flour 0.3%, corn steep liquor 4%, nitrate 0.075%, ammonium salt 0.075%, ammonia water 0.45%, phosphate 0.3%, sodium chloride 0.15%, magnesium sulfate 0.015%, zinc sulfate 0.015%, 0.05% defoamer, the balance is water; the induction medium, by weight percentage, inc...

Embodiment 3

[0033] A method for fermenting a high-content 12α-hydroxysteroid dehydrogenase fermented liquid, taking spare 12α-hydroxysteroid dehydrogenase expression gene recombinant E. Bacteria fermentation medium was cultivated, and induction medium was added after 2 hours for a total of 8 hours; the recombinant Escherichia coli expressing the 12α-hydroxysteroid dehydrogenase gene was a secondary fermentation strain, stored in glycerol tubes, and The fraction is 1-5% of the inoculum size, which is inserted into an Erlenmeyer shaker flask containing the LB liquid sterile medium, and placed in a shaking table at 37°C, 50RH%, and 240rpm for 12 hours for later use; the new sterile fermentation medium, Percentage by weight, including 0.5% corn flour, 6% corn steep liquor, 0.2% nitrate, 0.2% ammonium salt, 1% ammonia water, 0.5% phosphate, 0.5% sodium chloride, 0.05% magnesium sulfate, 0.05% zinc sulfate, 0.05% defoamer, the balance is water; the induction medium, by weight percentage, includ...

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Abstract

The invention provides a fermentation method for high-content 12[alpha]-hydroxysteroid dehydrogenase fermentation liquor, and belongs to the technical field of biology and medicines. the fermentationmethod comprises the following steps of: inoculating a spare recombinant escherichia coli containing a 12[alpha]-hydroxysteroid dehydrogenase expression gene into a novel sterile fermentation medium as a strain according to an inoculation amount of 2-6% of volume fraction to be cultured; and after 2h, carrying out fed-batch by a fed-batch induced medium totally for 8h. By use of the fermentation method, in a process that the spare recombinant escherichia coli containing the 12[alpha]-hydroxysteroid dehydrogenase expression gene is taken as the strain to ferment and produce the 12[alpha]-hydroxysteroid dehydrogenase (12[alpha]-HSDH), a high-nutrition low-cost novel medium is used for replacing a traditional LB (lysogeny broth) medium, the fed-batch induced medium is adopted, the bacteria OD600 (optical density) of culture bacteria is 50-60, and meanwhile, the enzyme activity of the 12[alpha]-hydroxysteroid dehydrogenase is 700-900U / L.

Description

technical field [0001] The invention relates to the technical field of biology and medicine, in particular to a fermentation method of a high-content 12α-hydroxysteroid dehydrogenase fermentation liquid. Background technique [0002] Enzymes are proteins or RNAs produced by living cells that are highly specific for their substrates and highly catalytically efficient. Enzymes are extremely important biocatalysts, they dominate many catalytic processes such as metabolism, nutrition and energy conversion of organisms, and most of the reactions closely related to life processes are enzyme-catalyzed reactions. [0003] Biocatalytic technology is a technology that uses enzymes or microbial cells or animal and plant cells as biocatalysts to catalyze reactions. As a biocatalyst, enzymes have many advantages over chemical catalysts: Enzyme-catalyzed reactions are generally carried out under normal temperature, normal pressure and near-neutral conditions, so they require less investm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N1/21C12R1/19
CPCC12N1/20C12N9/0006C12Y101/01176
Inventor 程玲利王婷婷田昌力龙柯利张翔黄清东吴眉平
Owner 四川澄华生物科技有限公司
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