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Method for semi-continuous fermentation culture of pichia pastoris engineering bacteria

A Pichia pastoris, fermentation and culture technology, applied in the field of bioengineering, can solve the problems of large material resource consumption, cumbersome procedures, low production efficiency and the like

Pending Publication Date: 2021-04-06
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Pichia pastoris belongs to induced fermentation. Looking at the current situation of the industry, it is mainly in the form of batch or fed-batch fermentation, which consumes a lot of human and material resources. Each batch of fermentation must be diluted, sterilized, inoculated, released Operations such as feeding and cleaning, the process is cumbersome, and the production efficiency is low. Therefore, how to design a fermentation mode with high fermentation efficiency is a technical point that needs to be solved urgently in this field.

Method used

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  • Method for semi-continuous fermentation culture of pichia pastoris engineering bacteria
  • Method for semi-continuous fermentation culture of pichia pastoris engineering bacteria
  • Method for semi-continuous fermentation culture of pichia pastoris engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] ①Use a 30L fermenter, inoculate 500ml of seed liquid into 10LBSM medium according to the inoculation ratio of 5%, stir at 300rpm, ventilate at 1vvm, tank pressure at 0.05MP, cultivate at 30°C and pH5.0 until the dissolved oxygen suddenly rises, and cultivate During the process, when the dissolved oxygen drops below 20%, the rotating speed is gradually increased to 700rpm.

[0045] ② Add 800ml of 50% glycerin (including 12ml / L PTM1), control the rate so that the dissolved oxygen is not lower than 20%, after the completion, take a sample to measure the wet weight of about 180g / L, and the whole process takes about 8 hours.

[0046] ③ Feed methanol (containing 12ml / L PTM1) for induction, control the rate so that the dissolved oxygen is not lower than 20%, and continue for 120h.

[0047] ④ After 120 hours of induction, release 50% of the feed liquid for harvesting, and add sterile BSM basic fermentation medium (without glycerol) to the fermenter until the wet weight is equal...

Embodiment 2

[0055] Wet weight, concentration and electrophoresis analysis were carried out on the samples during the fermentation process. The measurement method is: take 30ml of fermentation liquid from the sampling port of the fermentation tank, centrifuge at 10,000 rpm for 10 minutes with a centrifuge tube of known weight, wet weight: discard the supernatant and weigh, subtract the weight of the centrifuge tube from the weight to get the wet weight; concentration: take The protein concentration of the supernatant was determined by the G250 method; electrophoresis: electrophoresis analysis was carried out by SDS-PAGE Coomassie Brilliant Blue staining. The result is as Figure 1 ~ Figure 3 , Table 2 ~ Table 3 shown.

[0056] Depend on figure 1 , figure 2 , image 3 It can be seen that the wet weight, supernatant protein concentration, and target protein content of the four stages have the same change trend. Through three consecutive fermentation cycles, the fermentation time from th...

Embodiment 3

[0063] After the fermentation broth was harvested, the fermentation supernatant was purified using a nickel column, and the results were as follows:

[0064] Table 4: Summary of purification results

[0065]

[0066]

[0067] Comparison of production efficiency between semi-continuous fermentation and batch fermentation:

[0068] table 5

[0069]

[0070] The labor and energy consumption ratio of semi-continuous fermentation and batch fermentation:

[0071] Table 6

[0072] Fermentation type average labor average energy consumption average water consumption semi-continuous fermentation 1.25 people / day 25 degrees / day 2.5L / day batch fermentation 1.8 people / day 32 degrees / day 10L / day ratio 0.69:1 0.78:1 0.25:1

[0073] The results show that the semi-continuous fermentation cells are reused, the continuity is strong, the technological process is simplified compared with batch fermentation, and the work intensity and wor...

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Abstract

The invention relates to the field of bioengineering, and particularly relates to a method for semi-continuous fermentation culture of pichia pastoris engineering bacteria. According to the method, BSM is taken as a basic culture medium, methanol is taken as an inducer, continuous fermentation of pichia pastoris engineering bacteria and continuous expression of foreign proteins are carried out, inoculated bacteria are recycled, the continuity is high, the technological process is simplified compared with batch fermentation, the working intensity and workload are greatly reduced, and the yield of the foreign proteins is increased by 40% compared with batch fermentation. The method is simple in process, low in cost and suitable for industrial production.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for semi-continuous fermentation and cultivation of Pichia pastoris engineering bacteria. Background technique [0002] Fermentation engineering refers to the use of modern engineering technology to use certain specific functions of microorganisms to produce useful products for humans. It is generally divided into batch fermentation, fed-batch fermentation, and continuous fermentation. [0003] Pichia pastoris was used to produce single-cell protein (SCP) in the 1980s. It has a good fermentation basis and the density of bacteria can reach 100g / L dry weight. The components of the growth medium include inorganic salts, trace elements, biotin, nitrogen source and carbon source, which are cheap and non-toxic. It can grow rapidly in the medium with methanol as the only carbon source, and alcohol oxidase AOX, a key enzyme in the methanol metabolism pathway, can reach 30% of the ...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N1/16C12R1/84
CPCC12P21/00C12N1/16
Inventor 赵枭阳柴丹丹李婷李新乐张晓雷徐延伟赵巧辉李桂林付光宇吴学炜杨增利
Owner ZHENGZHOU IMMUNO BIOTECH
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