Dual-pump material feeding method and recombinant Escherichia coli fermentation method based on dual-pump material feeding method

A technology of recombinant Escherichia coli and fermentation method, applied in the field of microbial fermentation, can solve the problems of large dissolved oxygen fluctuation range and low fermentation level, and achieve the effects of large dissolved oxygen fluctuation range, improved fermentation level, and reduced dissolved oxygen fluctuation range

Active Publication Date: 2017-10-03
SHANDONG LUKANG PHARMA
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the present invention provides a double-pump feeding method and a method for fermenting recombinant Escherichia coli by using the double-pump feeding method, which solves the technical problems of large fluctuation range of dissolved oxygen and low fermentation level in the fermentation process of E. coli

Method used

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  • Dual-pump material feeding method and recombinant Escherichia coli fermentation method based on dual-pump material feeding method
  • Dual-pump material feeding method and recombinant Escherichia coli fermentation method based on dual-pump material feeding method
  • Dual-pump material feeding method and recombinant Escherichia coli fermentation method based on dual-pump material feeding method

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Inoculation: Take out the bacterial solution from the preserved glycerol tube and insert it into a shaker flask containing 150ml LB medium (containing ampicillin 500ug / ml) with a 1% inoculum amount, and culture it on a shaker at 37°C for 4 hours, and wait until the culture solution OD 600 When it grows to 2.5, it starts to be transplanted into the tank.

[0052] Prepare 10L fermentation medium according to the following formula: dodecahydrate and disodium hydrogen phosphate 200g, potassium dihydrogen phosphate 150g, ammonium chloride 200g, sodium sulfate 50g, magnesium sulfate heptahydrate 20g, peptone 200g, yeast powder 100g, glycerol 200g, Polyether type defoamer 10mL, ferric chloride 1.5g.

[0053] Tank elimination: Pour the prepared fermentation medium into the fermenter, and sterilize at 121°C for 30 minutes; after cooling down, control the fermentation temperature to 37°C, the ratio of ventilation to air is 1:1, the speed is 200rpm, and after 20 minutes of stabili...

Embodiment 2

[0060] Inoculation, tank elimination, and tank entry are the same as in Example 1.

[0061] Fermentation control: Fermentation tank parameters in the early stage of fermentation: 150rpm, 37°C, aeration ratio 1:0.5 (inlet air pressure 0.08MPa), pH of the fermentation broth is not controlled. With the fermentation broth OD 600 Growth, adjust the stirring speed of the fermenter and the air flow, so that the dissolved oxygen of the fermented liquid is controlled above 50%. When the pH starts to drop, add ammonia water to the fermentation broth, and adjust the pH of the fermentation broth to 6.5 in 0.5h; Supplemented with 60% w / v sterile glucose. The electronic switch of the feeding pump is set to be associated with the dissolved oxygen value, and the associated dissolved oxygen value is set to 25%, and the dissolved oxygen value of the fermented liquid is controlled to be 10-50%.

[0062] Induced expression: regular sampling to detect the OD of the fermentation broth 600 , to ...

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Abstract

The invention relates to the technical field of microbial fermentation and particularly relates to a dual-pump material feeding method and a recombinant Escherichia coli fermentation method based on the dual-pump material feeding method. The dual-pump material feeding method utilizes a constant-speed material feeding pump and a dissolved oxygen-related pump to supplement materials to the fermentation broth. The method comprises starting the constant-speed material feeding pump, adjusting a glucose flow-addition rate of the constant-speed material feeding pump to less than consumption rate of the glucose in the fermentation broth, starting the dissolved oxygen-related pump, setting a related dissolved oxygen value, when the dissolved oxygen value of the fermentation broth is higher than the preset value of the dissolved oxygen-related pump, starting the dissolved oxygen-related pump to supplement materials, and when the dissolved oxygen value of the fermentation broth is less than the preset value of the dissolved oxygen-related pump, stopping the dissolved oxygen-related pump. The dual-pump material feeding method realizes fermentation of recombinant Escherichia coli and shortens a dissolved oxygen fluctuation range to 10%. After fermentation, the OD value of the fermentation broth is 160. The enzyme activity of the hydrolase expressed by the recombinant escherichia coli in fermentation broth is 180 U / ml. The recombinant Escherichia coli fermentation method greatly improves the fermentation level of Escherichia coli.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a double-pump feeding method and a recombinant Escherichia coli fermentation method based on the double-pump feeding method. Background technique [0002] The fermentation process of bioengineering bacteria constructed by gene recombination technology is different from the traditional fermentation process. As far as the biological materials are selected, the former contains recombinant vectors with exogenous genes, while the latter is a single microbial cell; considering the fermentation process, the purpose of bioengineering bacteria fermentation production is to obtain a large amount of exogenous gene products, Minimize the contamination of the host cell's own protein. The high-level expression of exogenous genes not only involves the relationship between the host, vector and cloned gene, but also is closely related to the environmental conditions in which they ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/18C12R1/19
CPCC12N1/20C12N9/18
Inventor 史磊申洋郭强张勇郑长春
Owner SHANDONG LUKANG PHARMA
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