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High density fermentation method of glucose oxidase in pichia pastoris

A technology of glucose oxidase and high-density fermentation, applied in the field of microbial fermentation, can solve problems such as difficulty in purification, and achieve the effects of increasing secretion, reducing dosage and increasing GOD yield

Active Publication Date: 2018-04-20
河北省微生物研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The large-scale production of GOD widely adopts Aspergillus niger or Penicillium fermentation, but in the process of Aspergillus niger and Penicillium fermentation to produce GOD, the existence of a large number of foreign proteins such as catalase, cellulase and amylase brings considerable difficulties to the purification. difficulty

Method used

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  • High density fermentation method of glucose oxidase in pichia pastoris
  • High density fermentation method of glucose oxidase in pichia pastoris
  • High density fermentation method of glucose oxidase in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The strain was inoculated into the seed solution of the primary test tube of 3mL YPD medium according to the inoculum amount of 3%. After culturing for 12h at 30°C and 250 rpm, the strain was transferred to the shake flask of 500mL of 100mL BMGY secondary seed solution according to the inoculum amount of 3%. , 30°C, 250rpm after 12h culture, culture medium OD 600 To 10, obtain the culture expansion liquid of activated strain.

[0047] Inoculate in the fermentor of 10L by the inoculum of 10% inoculated in the fermentor that the bacterial classification expansion culture fluid of activation is filled, fermentor initially dresses 5.6LBSM medium. The initial fermentation control temperature was 28°C, the pH of the fermented liquid was controlled to be 5.0 with 28% concentrated ammonia water, and the dissolved oxygen on the tank was controlled at 0 by adjusting the stirring speed and ventilation. After the glycerin in the BSM medium is exhausted and the dissolved oxygen ris...

Embodiment 2

[0051] The strain was inoculated into the seed solution of the primary test tube of 3mL YPD medium according to the inoculum amount of 5%. After culturing for 16h at 30°C and 230 rpm, the strain was transferred to the shake flask of 500mL of 100mL BMGY secondary seed solution according to the inoculum amount of 5%. , 30°C, 230rpm cultured for 16h, the culture solution OD 600 To 10, obtain the culture expansion liquid of activated strain.

[0052] The bacterial classification expansion culture liquid of activation is inoculated in the fermentor of 10L by 9% inoculum size, and fermentor initially dresses 5.6LBSM substratum. The initial fermentation control temperature was 28°C, the pH of the fermented liquid was controlled to be 5.0 with 28% concentrated ammonia water, and the dissolved oxygen on the tank was controlled at 0 by adjusting the stirring speed and ventilation. After the glycerin in the BSM medium is exhausted and the dissolved oxygen rises, add 50% (w / v) glycerol c...

Embodiment 3

[0056] The strain was inoculated into the seed solution of the primary test tube of 3mL YPD medium according to the 8% inoculum amount. After culturing for 18 hours at 30°C and 200 rpm, the 8% inoculum amount was transferred to the 500mL 100mL BMGY secondary seed solution shake flask , 30°C, 200rpm after 18h culture, culture solution OD 600 To 10, obtain the culture expansion liquid of activated strain.

[0057] Inoculate in the fermentor of 10L by the inoculum of 10% inoculated in the fermentor that the bacterial classification expansion culture fluid of activation is filled, fermentor initially dresses 5.6LBSM medium. The initial fermentation control temperature was 28°C, the pH of the fermented liquid was controlled to be 5.0 with 28% concentrated ammonia water, and the dissolved oxygen on the tank was controlled at 0 by adjusting the stirring speed and ventilation. After the glycerin in the BSM medium is exhausted and the dissolved oxygen rises, add 50% (w / v) glycerol con...

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Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a high density fermentation method of glucose oxidase in pichia pastoris. According to the high density fermentation method of glucose oxidase in pichia pastoris, a pichia pastoris genetically engineered bacterium strain with a preservation number of CGMCC No.11626 is taken as an original strain, a mixture of methanol and sorbitol at a volume ratio of 20:1 is adopted for induction of fermentation, GOD yield is increased further, and discharge enzyme activity is capable of reaching 905U / ml.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a high-density fermentation method of glucose oxidase in Pichia pastoris. Background technique [0002] Glucose oxidase (Glucose oxidase, GOD, EC1.1.3.4) can catalyze the oxidation of β~D~glucose into δ~D~gluconic acid and hydrogen peroxide, and its function is to remove glucose, remove oxygen, form hydrogen peroxide, etc. aspect. As a natural biocatalyst, glucose oxidase is safe and has no side effects. It has many applications in food and beverage industry, livestock and poultry breeding industry, pharmaceutical industry and other fields, and has achieved considerable commercial value. GOD is used in bread, milk, juice, beer and other fields to deoxygenate and keep fresh; GOD added in animal feed can have anti-oxidation function, can significantly inhibit the production of moldy microorganisms, and has a good effect on moldy feed toxins Degradation, so as to im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12R1/84
CPCC12N9/0006C12Y101/03004
Inventor 高庆华王玥董聪王庆庆罗同阳刘蕾王云鹏
Owner 河北省微生物研究所有限公司
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