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Method for controlling Pichia pastoris to ferment to produce antibacterial peptide by utilizing dissolved oxygen parameters

A production method and parameter control technology, applied in the field of bioengineering, can solve the problems of no antibacterial peptide LL-37 genetically engineered yeast fermentation production method, etc., and achieve the effects of shortened antibacterial activity, simple preparation process, and reduced cost

Active Publication Date: 2011-02-23
山东华辰制药有限公司
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  • Abstract
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Problems solved by technology

There are few reports on the law of the fermentation process and patents on process control using parameter feedback. At the same time, so far, there is no domestic fermentation production method of the antibacterial peptide LL-37 genetically engineered yeast

Method used

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  • Method for controlling Pichia pastoris to ferment to produce antibacterial peptide by utilizing dissolved oxygen parameters
  • Method for controlling Pichia pastoris to ferment to produce antibacterial peptide by utilizing dissolved oxygen parameters
  • Method for controlling Pichia pastoris to ferment to produce antibacterial peptide by utilizing dissolved oxygen parameters

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Embodiment Construction

[0010] Technical scheme of the present invention is as follows:

[0011] A method for producing antimicrobial peptides by using dissolved oxygen parameters to control Pichia pastoris fermentation, characterized in that the steps are as follows:

[0012] (1) Add the culture medium into the fermenter, and after cooling it down, adjust the fermentation temperature to 27-30°C, calibrate the dissolved oxygen (DO ) to obtain the fermentation medium;

[0013] (2) Inoculate the activated gene yeast strain in the fermentation medium, the inoculation amount is 10% (v / v), mark it as 0 point at the same time, start fermentation, the fermentation conditions are: ventilation is 0.3~1.5vvm, stirring The speed is 100~400rpm, the tank pressure is 0.02~0.1MPa;

[0014] (3) When the DO value rises from less than 10% to more than 50%, start feeding methanol until the DO value falls back to 10% to 30%, stop feeding methanol, and when the DO value rises steadily to more than 50% again, Start fee...

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Abstract

The invention relates to a method for controlling Pichia pastoris to ferment to produce an antibacterial peptide by utilizing dissolved oxygen parameters, and belongs to the technical field of biological engineering. The method comprises the following steps of: (1) calibrating the percent of dissolved oxygen (DO) to obtain a fermentation medium; (2) simultaneously marking the percent as a 0 pointand starting to ferment; (3) when the value of the DO is up to above 50 percent from 10 percent below, starting to feed methanol until the value of the DO falls back to 10 to 30 percent; stopping feeding the methanol; when the value of the DO is stably up to over 50 percent again, starting to feed the methanol again until the value of the DO falls back to 10 to 30 percent; stopping feeding the methanol; operating circularly; and controlling the value of the DO to be maintained between 10 and 30 percent all the time by feeding the methanol, adjusting ventilation and controlling a rotation speed; and (4) when the value of the DO does not fall back after feeding the methanol, finishing fermenting. The method of the invention has a short fermentation period and less dosage of the methanol; and compared with conventional dissolved oxygen value regulation and control, the method is easier to operate, and conditions are easier to grasp.

Description

technical field [0001] The invention relates to a method for controlling Pichia pastoris fermentation to produce antimicrobial peptides by utilizing dissolved oxygen parameters, and belongs to the technical field of bioengineering. Background technique [0002] Antibacterial peptides (antibacterial peptides, ABP) are a kind of defensive peptide active substances produced by the innate immune system of multicellular eukaryotes under the condition of exogenous pathogen induction, and their molecular weight is generally 2-5ku. Compared with traditional antibiotics, antimicrobial peptides are less likely to make bacteria resistant. [0003] In recent years, the study of yeast as the recipient of genetic engineering has attracted people's attention. Yeast has a more complete gene expression regulation mechanism and the ability to process, modify and secrete expressed products than E. coli, and it does not produce endotoxin. Good eukaryotic gene recipient bacteria in engineering....

Claims

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Application Information

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IPC IPC(8): C12R1/84C12P21/00
Inventor 姜正军陈溥言袁永苏晓明朱孟豪田建国王希凤孙大明
Owner 山东华辰制药有限公司
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