Method for high-yield bacterial strains by high-flux screening of gentamycin based on ARTP composite mutagenesis technique

A technology of gentamicin and high-yielding strains, applied in the field of microbial culture, can solve the problems of low increase in gentamicin production capacity, lack of a complete set of gentamicin strains, and single screening method, achieving obvious mutagenic effects , increased probability, and convenient operation

Inactive Publication Date: 2019-11-26
QINGDAO INNOVATION INST OF EAST CHINA UNIV OF SCI & TECH +1
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Problems solved by technology

[0003] Most of the strains of Micromonas crimson rubrum or Micromonas aculeatus that produce gentamicin in actual enterprises are obtained by traditional screening methods, such as shaking flask culture, liquid phase or biological method to detect titer, which has high investment and long screening period. T...

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  • Method for high-yield bacterial strains by high-flux screening of gentamycin based on ARTP composite mutagenesis technique
  • Method for high-yield bacterial strains by high-flux screening of gentamycin based on ARTP composite mutagenesis technique

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Embodiment

[0040] A method for high-throughput screening of high-yield strains of gentamicin based on ARTP compound mutagenesis technology, the flow chart is as follows figure 1 shown, including the following steps:

[0041] The first step, preparation of spore suspension: use an inoculation shovel to scrape the mature spores on the culture medium from the spores or slant and eggplant bottle preserved in the glycerol tube and transfer them to a centrifuge tube containing 10ml sterile saline to prepare the spore suspension solution, the prepared spore suspension was prepared at a concentration of 2×10 9 number of spores, and then use physiological saline dilution gradient 3 gradients of 10 -4 、10 -5 and 10 -6 Carry out ARTP compound mutagenesis treatment.

[0042] The second step, ARTP compound mutagenesis treatment and cultivation: adding 500ul of lithium chloride solution with a concentration of 10% to 500ul of which the number of spores is 10 5 1 / ml single spore suspension, shake ...

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Abstract

The invention relates to a method for high-yield bacterial strains by high-flux screening of gentamycin based on an ARTP composite mutagenesis technique. The method comprises the steps of preparing aspore suspension, performing ARTP mutagenesis composite mutagenesis, performing quick detection on products, prescreening high-yield bacterial strains with gentamycin and performing screening once again to obtain high-yield bacterial strains and performing stability inspection on the high-yield bacterial strains. Compared with the prior art, the method has the advantages that the principle that OPA and the gentamycin are subjected to a reaction to form characteristic absorption peaks of about 330nm is adopted, through a microplate reader, quick determination of an OD value is realized, so thatfast detection of the gentamycin can be successfully realized. The interference problems that poor specificity of a phosphotungstic acid method and a method for forming precipitate with the gentamycin to cause reduction of the OD value, fermentation liquid color and the like, are solved. The method has the characteristics of being quick, high-flux, convenient to operate, accurate and the like, and the mutation breeding and screening efficiency of the gentamycin is improved to a great extent, so that the probability for obtaining the high-yield bacterial strain with the gentamycin is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of microorganism culture, and in particular relates to a method for high-throughput screening of high-yielding gentamicin strains based on ARTP compound mutagenesis technology. Background technique [0002] Gentamicin is an aminoglycoside antibiotic produced by fermentation and extraction of micromonas (micromonas crimson or micromonas aculeatus), which can inhibit the biosynthesis of bacterial proteins, thereby inhibiting Bactericidal effect, has a strong bactericidal effect on Gram-negative bacteria and some Gram-positive bacteria. Gentamicin is the main aminoglycoside antibiotic, and its market accounts for 50% of aminoglycoside antibiotics. Therefore, it is of great significance for practical application to develop high-throughput screening mutagenesis for gentamicin industrial production strains to obtain high-yielding strains. [0003] Most of the strains of Micromonas crimson rubrum or Micromonas ac...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12Q1/04
CPCC12N15/01C12Q1/04
Inventor 李敏超李兰张嗣良庄英萍储炬杭海峰
Owner QINGDAO INNOVATION INST OF EAST CHINA UNIV OF SCI & TECH
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