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Process for transforming antibacterial peptide AD gene by yeast fermentation

A technology of gene transformation and yeast fermentation, applied in biochemical equipment and methods, fermentation, genetic engineering and other directions, can solve the problems of fermentation production methods without genetically engineered bacteria, and achieve the effects of low cost, stable quality and short production cycle

Inactive Publication Date: 2004-10-27
广州市微生物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And so far, there is no fermentation production method of this genetically engineered bacterium in China.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The production of the antimicrobial peptide of embodiment 1 once 50 liters tanks

[0017] 1. Shake flask culture

[0018] 50 liters of fermenter, the capacity is 30 liters, if the inoculum size is 5%, the amount of culture medium for the shake flask is 30 liters × 5% = 1.5 liters, according to 0.8% yeast powder, 2% peptone, 1% glucose, pH Formula 6.8, with 1.5 liters of culture medium, divided into 500 ml Erlenmeyer flasks, the volume of each bottle is 100 ml, sterilized after filling, cooled to 30°C and inserted into the strain (about 3 ml / bottle), shaker The rotating speed is 180 rpm, the temperature is controlled at 30°C, and after 36 hours of cultivation, it is inserted into a 50-liter fermenter for cultivation.

[0019] 2. Fermenter culture

[0020] The actual capacity of 50 liters of tanks is 30 liters, minus 1.5 liters of shake bottle seeds, then the actual distribution is 30-1.5=28.5 liters, and the cultivation of 28.5 liters is based on the 50 liters of tanks...

Embodiment 2

[0021] The production of the antimicrobial peptide of embodiment 2 once 500 liters tanks

[0022] 1. Shake flask culture

[0023] 50 liters of fermenter, with a capacity of 30 liters, assuming that the inoculum size is 10%, the amount of medium for shaker flasks is 30 liters × 10% = 3 liters, according to yeast powder 1%; peptone 1.5%; glucose 1.5%; pH Formula 7, with 3 liters of culture medium, divided into 500 ml Erlenmeyer flasks, the volume of each bottle is 100 ml, sterilized after packing, cooled to 28 ° C to insert bacteria (about 3 ml / bottle), shaker The rotating speed is 180 rev / min, and the control temperature is 28°C. After 36 hours of cultivation, it is inserted into a 50-liter seed tank for cultivation.

[0024] 2. Seed tank cultivation

[0025] 50 liters of tank actual capacity is 30 liters, minus 3 liters of shake bottle seeds, then the actual distribution is 30-3=27 liters, the cultivation of 27 liters is based on 50 liters of tanks (actual The water capacit...

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Abstract

A process for preparing the AD gene transformed antibacterial peptide yeast by fermentation features that the antibacterial peptide contained engineering yeast obtained by transforming AD gene plasmid is fermented in proper culture medium and culture condition to express the antibacterial peptide gene and secrete the antibacterial peptide outside the thallus. The sterilizing activity of antibacterial peptide in the fermented liquid can reach 7000 unit / ml. Its advantages are high E / C ratio, and low cost.

Description

technical field [0001] The invention belongs to the field of biological fermentation, and more specifically relates to an antimicrobial peptide AD gene transformation yeast fermentation production method. Background technique [0002] The antimicrobial peptide AD gene is an artificially synthesized gene designed according to the amino acid sequence of the tussah silkworm natural antimicrobial peptide A and antimicrobial peptide D genes. Patent 01112278.1 discloses a technique of cloning the 1-11 amino acid sequence of tussah silkworm natural antimicrobial peptide A and the 12-37 amino acid sequence of antimicrobial peptide D into Pichia pastoris. The genetically engineered yeast transformed with the plasmid containing the antimicrobial peptide AD gene is obtained by cloning the natural antimicrobial peptide A and antimicrobial peptide D genes of tussah silkworm into yeast. Due to the unique bactericidal mechanism of antimicrobial peptides, pathogenic bacteria are not easy t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N1/19C12N15/12C12N15/81C12P19/34
Inventor 彭中健杨冠东杜少平梁淑娃
Owner 广州市微生物研究所
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