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Composite mutagenesis method of aspergillus niger

A technology of compound mutagenesis and Aspergillus niger, which is applied in the field of mutagenesis and breeding of microbial strains, can solve problems such as DNA damage, and achieve the effects of convenient operation, good genetic stability and high efficiency

Inactive Publication Date: 2017-05-24
JIANGSU GUOXIN UNION ENERGY CO LTD
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Problems solved by technology

In addition, the reactive oxygen species (ROS) generated by the plasma enter the cell due to the enhanced permeability of the cell membrane, which further causes DNA damage.

Method used

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  • Composite mutagenesis method of aspergillus niger

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Embodiment

[0028] A compound mutagenesis method of Aspergillus niger, described method is to carry out normal pressure room temperature plasma mutagenesis on Aspergillus niger protoplast, specifically comprises the steps:

[0029] (1) Spore pre-cultivation, transfer the glycerin tube of Aspergillus niger frozen at -80°C to the slant medium (malt extract 30g / L, tryptone 5g / L, agar 1.5%, sterilized at 121°C for 15min) Activation culture, cultured at 37°C for 6 days, after activation twice, scraped spores; 6 Inoculate the inoculation amount of each / mL into a 250mL Erlenmeyer flask filled with 50mL of medium, culture at 30°C and 200rpm for 16h, and then collect the balls;

[0030] (2) Bacterial enzymes to detach the wall: take the bacterium ball and wash it with KMC solution for 3 times, then add 50mL lysozyme and chitinase solution (5mg / mL lysozyme, 0.2U / mL chitinase, 0.22 μM sterile filter), mix well, place in a 37°C water bath for 2 hours, centrifuge at 2000g for 10 minutes, discard the ...

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Abstract

The invention discloses a composite mutagenesis method of aspergillus niger. The method comprises the following steps of: (1) pre-culturing spores; (2) carrying out enzymolysis on thalli and removing cell walls; (3) carrying out normal-pressure and room-temperature plasma mutagenesis; (4) carrying out regeneration culture on protoplasts; (5) screening by a 24-pore plate; and (6) fermenting by a shake flask and screening again. According to the method disclosed by the invention, the protoplasts without the cell walls are adopted and a mutagenic agent more easily enters cell nucleuses without the blocking of the cell walls and can rapidly act on hereditary substances; and the probability that the hereditary substances are mutated is greater and the efficiency is higher; and the mutagenesis effect is more obvious than a predicated effect.

Description

technical field [0001] The invention relates to the technical field of mutagenesis and breeding of microbial strains, in particular to a method for compound breeding of Aspergillus niger citric acid high-yielding strains by combining protoplasts with normal pressure and room temperature plasma mutagenesis. Background technique [0002] Citric acid has broad application prospects and market demand. For economical considerations, some studies use the cheaper raw sugar raw materials and agricultural waste residues to produce citric acid on the basis of existing strains, while the other part of research is devoted to improving strains. The transformation and selection of production strains are the basis of citric acid fermentation industry, which determines the success or failure of the fermentation process and the value of industrial production of fermentation products. my country is a big country for citric acid production. Although the conversion rate of citric acid is close...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12R1/685
CPCC12N15/01C12N13/00
Inventor 彭艳红胡志杰蒋小东孙福新金赛邵玉芬李江华刘龙
Owner JIANGSU GUOXIN UNION ENERGY CO LTD
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