Compound induction mutation method for high-producing strain capable of producing epsilon-poly-L-lysine at high yield

A technology of high-yield strains and compound mutagenesis, applied in the field of fermentation engineering, can solve the problems of low natural mutation rate of microbial strains, achieve the effects of increasing genetic stability, increasing damage, and simple operation methods

Inactive Publication Date: 2017-03-22
SICHUAN ACAD OF FOOD & FERMENTATION INDS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Microorganisms are closely related to the brewing industry, food industry, and biological product industry. The quality of their strains is directly related to the quality of various industrial products. The natural mutation rate of microbial strains is very low. Well-bred strains are far from meeting people's needs, so it is necessary to cultivate high-quality, high-yielding microbial strains

Method used

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  • Compound induction mutation method for high-producing strain capable of producing epsilon-poly-L-lysine at high yield
  • Compound induction mutation method for high-producing strain capable of producing epsilon-poly-L-lysine at high yield

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Experimental program
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Effect test

Embodiment 1

[0035] Mutagenesis with cobalt-60 radiation

[0036] 1.1 Take the commercially available ε-poly-L-lysine-producing Streptomyces griseus as the starting strain, pick a ring and inoculate it on a fresh slant medium, and culture it in a constant temperature incubator at 30.5°C for 120 hours to activate and rejuvenate;

[0037] 1.2. Cultivate fresh slopes rich in spores, select four, and irradiate with cobalt-60 rays. The cumulative radiation doses are 40,000 rad, 60,000 rad, 80,000 rad, and 100,000 rad respectively;

[0038] 1.3. For the inclined surfaces of the four strains after irradiation (different radiation doses), pick a ring and put them into a sterilized Erlenmeyer flask filled with 20mL distilled water and glass beads (diameter 4mm, 25 pieces), shake for 30min, and prepare bacterial suspension.

[0039] 1.4. The prepared bacterial suspension was diluted with five gradient concentrations (10 -1 、10 -2 、10 -3 、10 -4 、10 -5 ), draw 0.1mL of different concentrations t...

Embodiment 2

[0048] First mutagenesis with cobalt-60 radiation

[0049] 1.1 Take the commercially available ε-poly-L-lysine-producing Streptomyces griseus as the starting strain, pick a ring and inoculate it on a fresh slant medium, and culture it in a constant temperature incubator at 30.5°C for 120 hours to activate and rejuvenate;

[0050] 1.2. Cultivate fresh slopes rich in spores, select four, and irradiate with cobalt-60 rays. The cumulative radiation doses are 40,000 rad, 60,000 rad, 80,000 rad, and 100,000 rad respectively;

[0051] 1.3. For the inclined surfaces of the four strains after irradiation (different radiation doses), pick a ring and put them into a sterilized Erlenmeyer flask filled with 20mL distilled water and glass beads (diameter 4mm, 25 pieces), shake for 30min, and prepare bacterial suspension.

[0052] 1.4. The prepared bacterial suspension was diluted with five gradient concentrations (10 -1 、10 -2 、10 -3 、10 -4 、10 -5 ), draw 0.1mL of different concentrat...

Embodiment 3

[0071] When the commercially available epsilon-poly-L-lysine-producing Streptomyces griseus was directly used for ARTP mutagenesis, it was found that the yield of the strain was about 12 g using the single method of ARTP, and the yield was not further improved by continuing to use this method. At the same time, the ARTP single method is frequently used for mutagenesis. Because of the high radiation energy level, after three generations, the spore-producing ability of the strain will decrease, which will affect the performance of the strain.

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Abstract

The invention belongs to the field of fermentation engineering and particularly relates to a compound induction mutation method for a high-producing strain capable of producing epsilon-poly-L-lysine at a high yield. The method comprises the following steps: performing initial screening based on fatality rate, separation, purification and diluting times, single colony site condition, developing and transparent ring size on a resistant plate by irradiation of cobalt 60 rays with different radiant quantities; then coating the plate when the accumulated radiant quantity is 80 thousand rad, the fatality rate is 90-95%, and the diluting concentration is 10<-4>-10<-5>; selecting single colonies which are big in colony size, develop color and obviously relatively big in transparent ring on the resistant plate for bottle shake, and performing culturing in a 10L fermenting tank for screening; and finally obtaining the high-producing strain, the output of which reaches 12g/L. Compared with that (9g/L) of an original strain, the output is increased by 33% or above. A passage stability test verifies that the strain is good in passage stability. By virtue of an ARTP induction mutation method, the high-producing strain, the output of which reaches 17g/L is finally obtained, and the strain is good in passage stability.

Description

technical field [0001] The invention belongs to the field of fermentation engineering, and in particular relates to a compound mutagenesis method for high-yield ε-poly-L-lysine strains. Background technique [0002] ε-poly-L-lysine is a lysine homopolymer containing 25-30 residues, it is easily soluble in water, but insoluble in organic solvents such as ethyl acetate, ethanol, ether; its thermal stability High resistance, 120 ℃ heating still has antibacterial activity. ε-poly-L-lysine is a polypeptide with antibacterial effect, and is considered to be a microbial food preservative with excellent antiseptic performance and great commercial potential among natural preservatives. Because of its broad-spectrum antibacterial properties, it has a strong inhibitory effect on most Gram-positive bacteria, Gram-negative bacteria and fungi, and its antibacterial effect is significantly stronger than that of α-polylysine, and it is safe With the advantages of high performance, good w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/01C12P13/02C12R1/545
CPCC12N15/01C12N1/20C12P13/02
Inventor 王刚万玉军唐晓芳李南臻李镇江庹有朋
Owner SICHUAN ACAD OF FOOD & FERMENTATION INDS
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