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Fermentation process for regulating pH and improving production level of recombinant human collagens

A human-derived collagen and fermentation process technology, which is applied in the field of fermentation technology by adjusting pH to improve the production level of recombinant human-derived collagen, can solve the problems of low production efficiency, long fermentation cycle, low protein expression and fermentation level, etc. The effect of improving biosynthesis rate, improving fermentation level and saving production cost

Active Publication Date: 2018-05-18
JIANGSU JLAND BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional process, the fermentation process of genetically engineered bacteria of collagen has the advantages of easy availability of raw materials, environmental protection, and stable product quality, but there are also problems such as longer fermentation cycle and lower production efficiency.
Chinese patent 201010602214.8 discloses a production method for expressing recombinant human-like collagen in Pichia pastoris. Pichia pastoris C13 is used as the strain, the expression level of recombinant collagen is 5g / L, the fermentation period is 99 hours, and the fermentation level 0.051g / L·h, although the fermentation cycle is short, the protein expression and fermentation level are too low
Chinese patent 201110327865.5 constructed a Pichia pastoris genetically engineered strain of recombinant human collagen. The genetically engineered strain was fermented to obtain recombinant human collagen. The fermentation period was 136 hours, and the protein expression was 16g / L. The level is 0.118g / L·h, although the protein expression has increased, but the fermentation period is too long and the fermentation level is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerin 40.0g / L; PTM1 4.35mL / L.

[0017] Add the seed liquid to the 1000L fermenter containing 500L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, the air flow and the speed are adjusted to make dissolved oxygen (DO) > 30%, and the initial stage control The pH is 5.2. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerin was added to supplement the carbon source until the wet weight of the bacteria was 214g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 18 hours. After the glycerol was exhausted, the pH was controlled to be 5.0, and methanol was started to flow into the stage of methanol induction culture. Adjust the rotation speed, air flow rate and feed methanol spe...

Embodiment 2

[0019] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerol 40.0g / L; PTM1 4.35mL / L.

[0020] Add the seed liquid to the 1000L fermenter containing 500L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, the air flow and the speed are adjusted to make dissolved oxygen (DO) > 30%, and the initial stage control The pH is 5.2. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerin was added to supplement the carbon source until the wet weight of the bacteria was 214g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 18 hours. After the glycerol was exhausted, the pH was controlled to 4.8, and methanol was started to be fed into the methanol-induced culture stage. Adjust the rotation speed, air flow rate and feed methanol speed to...

Embodiment 3

[0022] Fermentation medium: 85% H 3 PO 4 26.7mL / L; CaSO 4 2H 2 O 1.175g / L; K 2 SO 4 18.2g / L; MgSO 4 ·7H 2 O 14.9g / L; KOH 4.13g / L; glycerol 40.0g / L; PTM1 4.35mL / L.

[0023] Add the seed liquid to the 1000L fermenter containing 500L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, the air flow and the speed are adjusted to make dissolved oxygen (DO) > 30%, and the initial stage control The pH is 5.3. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerin was added to supplement the carbon source until the wet weight of the bacteria was 214g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 18 hours. After the glycerol was exhausted, the pH was controlled to 4.8, and methanol was started to be fed into the methanol-induced culture stage. Adjust the rotation speed, air flow rate and feed methanol speed to...

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Abstract

The invention discloses a fermentation process for regulating pH and improving a production level of recombinant human collagens. The process comprises the following steps: inoculating pichia pastorisculture liquid into a sterile fermentation medium, controlling the pH value to be 5.2-5.4 at the initial growth phase, fermenting and culturing for 14-18 hours, regulating the pH value to 4.8-5.0, and supplementing methanol to perform induced expression. According to the process disclosed by the invention, in the fermentation process, the pH value is reduced at the methanol induced expression stage, strain growth is controlled, the biosynthesis rate of the recombinant human collagens is improved, the biosynthesis rate of the recombinant human collagens can be further improved, and the fermentation time is shortened. Meanwhile, the expression quantity of the recombinant human collagens is increased, the fermentation level is improved by 20% or higher, the production cost is saved, and particularly the process is applicable to large-scale industrialized production of the recombinant human collagens and can bring huge actual application value for the industrialized production.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation and relates to a fermentation process for adjusting pH to increase the production level of recombinant human collagen. Background technique [0002] Collagen is an important protein in animals. It is widely distributed in tissues such as skin, cartilage, and blood vessels. It participates in cell migration, differentiation, and reproduction, and plays an important role in maintaining the normal physiological functions of cells, tissues, and organs. It is used in food , Feed, beauty, cosmetics, medicine and other fields are widely used. At present, collagen raw materials are mainly obtained by separation and purification after treating animal skin, bone and other tissues such as pigs and cattle with physical and chemical methods such as acid, alkali and heating. However, the collagen obtained by the above method has complex components and poor water solubility, and because it comes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/84
CPCC07K14/78C12P21/02
Inventor 杜尔凤黄建民季乐赵健烽冯丽萍陶海
Owner JIANGSU JLAND BIOTECH CO LTD