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Method for producing glucose oxidase via fermentation

A technology for glucose oxidase and strain production, applied in the field of genetic engineering, can solve the problems of low enzyme activity, increased production cost, difficult separation and purification work, etc., and achieves the effect of improving enzyme activity

Active Publication Date: 2013-02-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Low yield, low enzyme activity, and complex detection methods are the limiting factors for the industrialization of GOD
Aspergillus niger or Penicillium fermentation is widely used in the large-scale production of glucose oxidase, but the fermentation of Penicillium and Aspergillus niger produces various by-products such as catalase, cellulase, amylase, etc., and a large amount of miscellaneous proteins are very important for the separation and purification in the later stage. The work creates great difficulties and also increases production costs

Method used

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  • Method for producing glucose oxidase via fermentation
  • Method for producing glucose oxidase via fermentation
  • Method for producing glucose oxidase via fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The 3L fermentation tank fermentation of embodiment 1 recombinant bacterium

[0022] The Pichia pastoris GS115-pPIC9K-GOX constructed in the previous work of our laboratory was used as the production strain. After activation, a single colony was picked and inoculated in 50mL YPD (50mL medium / 500mL Erlenmeyer flask), and heated at 30°C, 200r / min Cultivate for 24h. The bacterial liquid in YPD was introduced into a 3L automatic fermenter (LiFlusGM BioTRON, Korea), with an inoculum volume of 10%, pH 5.5 was controlled with 25% ammonia water, the temperature was 30°C, and the stirring speed and ventilation were adjusted to maintain dissolved oxygen above 30%. When the glycerol was exhausted (DO rose rapidly), the growth medium was fed with 50% glycerol. When the bacteria reached 100g / L, stop feeding. After the glycerol was depleted again and the substrate was deprived for about 1 hour, the induction medium was fed, the induction temperature was lowered to 22°C, and the mix...

Embodiment 2

[0030] 3L fermenter fermentation of embodiment 2 recombinant bacteria

[0031] Other conditions are the same as in Example 1, only the induction conditions are changed, the induction temperature is reduced to 20°C, the mixed addition ratio of methanol and sorbitol is controlled to be 10:2 (W / W) and the concentration of methanol in the whole induction process is maintained at 2.5% (W / V), inducing the expression of glucose oxidase.

[0032] The highest enzyme activity of the expressed glucose oxidase was 600U / mL in a 3L fermenter, the highest dry weight was 231.6g / L, and the highest specific enzyme activity was 82452U / g.

Embodiment 3

[0033] The 3L fermentation tank fermentation of embodiment 3 recombinant bacteria

[0034] Other conditions are the same as in Example 1, only the induction conditions are changed, the induction temperature is reduced to 25°C, the mixed addition ratio of methanol and sorbitol is controlled to be 10:1 (W / W) and the concentration of methanol in the whole induction process is maintained at 2.0% (W / V), inducing the expression of glucose oxidase.

[0035] The highest enzyme activity of the expressed glucose oxidase was 615U / mL in a 3L fermenter, the highest dry weight was 234.2g / L, and the highest specific enzyme activity was 83255U / g.

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Abstract

The invention discloses a method for producing glucose oxidase via fermentation, belonging to the field of fermentation engineering. According to the method, a recombined pichia pastoris GS115-pPIC9K-GOX genetically engineered bacterium which is capable of producing glucose oxidase with relatively high activity and constructed in the preliminary work in the laboratory is used; and the collection number of the genetically engineered bacterium is CCTCC NO: M2012266. At the methanol induction state, the enzyme activity of the glucose oxidase is improved through adding sorbitol, namely, controlling the residual concentration 1.8% (W / V) of the methanol in the fermentation liquor; and when the mixed addition proportion of the methanol to the sorbitol is 10: 1 (W / V), the enzyme activity of the glucose oxidase is 645.3U / mL which is improved by 50.9% relative to the enzyme activity (427.6U / mL) of not adding the sorbitol, and the fermentation period is effectively shortened, so that a favorable foundation is laid for the large-scale production of the glucose oxidase.

Description

technical field [0001] The invention relates to a method for fermenting and producing glucose oxidase, which belongs to the field of genetic engineering. Background technique [0002] Glucose oxidase (β-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4, referred to as GOD), is a glycosyl-rich lutein, which can specifically catalyze β -D-glucose produces gluconic acid and hydrogen peroxide. [0003] Glucose oxidase (GOD) is one of the most important tool enzymes in the biological field. Since Updike and Hicks immobilized GOD on the surface of Clark oxygen electrode and applied it to blood glucose measurement in 1967, GOD has been widely used in food, feed, medicine and many other related fields. [0004] In the food industry, the presence of oxygen causes many chemical reactions that are not conducive to product quality and creates conditions for the growth of many microorganisms. At present, many countries have widely used GOD as a safe antioxidant for workers in various fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12R1/84
Inventor 张娟陈坚顾磊堵国成沈伊娜李婷李梦洁殷政
Owner JIANGNAN UNIV