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Fermentation process to improve the production level of recombinant human collagen and reduce the protein degradation rate

A human-sourced collagen, production-level technology, applied in animal/human protein, fermentation, microbial-based methods, etc., can solve the problems of low protein expression and fermentation level, long fermentation cycle, low production efficiency, etc., and achieve improved The effect of expression amount, mitigation of degradation, and production cost saving

Active Publication Date: 2022-05-13
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, and the recombinant collagen expression is 5g / L. The level is 0.051g / L·h, although the fermentation period is short, but 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
And in actual production, the product degrades at the end of fermentation, and some batches even degrade to 3g / L

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Fermentation medium is basal salt 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.

[0019] Add the seed solution into a 10L fermenter containing 6L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, adjust the air flow and speed to make dissolved oxygen (DO) > 30%, during the fermentation process Control pH5.2. 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 215g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 16 hours. After the glycerol was exhausted, methanol was added to enter the stage of methanol induction culture, and ammonium citrate was added at one time at a rate of 0.1 g / L. Dissolved oxygen (DO) > 20% b...

Embodiment 2

[0021] Fermentation medium is basal salt 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.

[0022] Add the seed solution into a 10L fermenter containing 6L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, adjust the air flow and speed to make dissolved oxygen (DO) > 30%, during the fermentation process Control pH5.0. 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 215g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 16 hours. After the glycerin was exhausted, methanol was added to enter the stage of methanol induction culture, and ammonium citrate was added at one time at a rate of 10 g / L. Dissolved oxygen (DO) > 20% by...

Embodiment 3

[0024] Fermentation medium is basal salt 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.

[0025]Add the seed solution into a 10L fermenter containing 6L fermentation medium according to the inoculation amount of 10%, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, adjust the air flow and speed to make dissolved oxygen (DO) > 30%, during the fermentation process Control pH5.2. 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 215g / L, and the glycerin supplement was stopped. At this time, the fermentation and cultivation time was 16 hours. After the glycerol was exhausted, methanol was added to enter the stage of methanol induction culture, and ammonium citrate was added at one time at a rate of 2.0 g / L. Dissolved oxygen (DO) > 20% by...

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Abstract

The invention discloses a fermentation process for improving the production level of recombinant human collagen and reducing the protein degradation rate. The process firstly inoculates Pichia pastoris seed liquid into a sterilized fermentation medium for fermentation, and adds 0.1-10 g / L ammonium citrate to the fermentation medium in the methanol induction culture stage. In the fermentation process of the present invention, the ammonium citrate is selected to be added in the methanol-induced expression stage to increase the biosynthesis rate of the recombinant human collagen, and the continuous feeding method can further increase the biosynthesis rate of the recombinant human collagen , the fermentation time is shortened, and the expression of recombinant human collagen is increased at the same time, and the fermentation level is increased by more than 20%. At the same time, the stability of recombinant human collagen in the fermentation broth is also improved, which saves production costs, and is especially suitable for recombinant human collagen. The industrialized large-scale production of human collagen can bring huge practical application value to industrialized production.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation and relates to a fermentation process for improving the production level of recombinant human collagen and reducing the protein degradation rate. 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 solub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C07K14/78C12R1/84
CPCC12P21/02C07K14/78
Inventor 杜尔凤陶海许乔林赵健烽黄建民
Owner JIANGSU JLAND BIOTECH CO LTD
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