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Mutation breeding method of gamma-PGA high-yield strain

A strain and high-yield technology, applied in biochemical equipment and methods, mutant preparation, treatment of microorganisms with electricity/wave energy, etc., can solve the problems of limited increase in strain yield, achieve high mutagenesis efficiency, simple mutagenesis operation, The effect of avoiding the saturation effect

Pending Publication Date: 2020-11-03
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a simple and practical, high-mutation range, high-throughput breeding method to solve the problem that the current gamma-PGA production strain mutagenesis selection method has a limited increase in strain yield

Method used

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  • Mutation breeding method of gamma-PGA high-yield strain
  • Mutation breeding method of gamma-PGA high-yield strain
  • Mutation breeding method of gamma-PGA high-yield strain

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Experimental program
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Effect test

Embodiment 1

[0042] The mutagenesis method of the high-yielding bacterial strain of γ-PGA comprises the following steps.

[0043] Mutagen formulation.

[0044] NTG mother liquor: Weigh 0.4 g of NTG, add a small amount of acetone to dissolve it, transfer it into a 10mL volumetric flask and make it to volume, and store it in a dark place and refrigerated.

[0045] DES mother solution: take 0.5mL of DSE liquid, add ethanol to aid dissolution, use phosphate buffer to make up to 10mL, and store in a dark place and refrigerated.

[0046] 5-BrU mother solution: Weigh 25 mg of 5-BrU and add 10 mL of sterile water, and store in the dark and refrigerated.

[0047] When taking the above solution, use a 0.22 μm sterile filter head to filter and sterilize, and use it after dilution.

[0048] Phosphate buffered saline (PBS): Solution A (0.2 mol / L Na 2 HPO 4 ): weigh Na 2 HPO 4 .12H 2O 71.6 g, add water and stir to dissolve, add water to make up to 1 L, autoclave at 121 ℃ for 20 min. Prepare moth...

Embodiment 2

[0060] The screening of high-yield gamma-PGA strains comprises the following steps.

[0061] (1) Primary screening of bacterial strains: select a plate with a lethal rate of 80-95%, and measure the size of the transparent circle (see figure 2 ), select the strain with larger K value.

[0062] K=d / D; d: diameter of colony; D: diameter of transparent circle.

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Abstract

The invention discloses a method for efficient mutation and rapid screening of gamma-PGA. Firstly, an original strain is subjected to compound mutation through diethyl sulfate, nitrosoguanidine, ultraviolet light and 5-brouracil, a strain C2 with high yield and high stability is obtained after screening and primary screening through a transparent ring method, secondary screening through a 24-poreplate and passage stability testing, and the polyglutamic acid yield of the strain reaches 21.3 g / L and is increased by 43.91% compared with that before mutation. The diethyl sulfate, the nitrosoguanidine, the ultraviolet light, the 5-brouracil and other mutagenic agents are combined for use, so that the mutation amplitude of the strain is remarkably improved. In combination with a perforated plate technology, rapid screening of polyglutamic acid mutant strains is realized.

Description

technical field [0001] The invention belongs to the field of microbial breeding of bioengineering, and relates to a method for breeding mutant strains using poly-γ-glutamic acid (γ-PGA). According to the method, high-yield γ-PGA fermentation strains can be quickly obtained. Background technique [0002] With the rise of bio-based polymer materials, γ-PGA has attracted great interest. γ-PAG is mostly produced by Bacillus and is an anionic heteromorphic peptide. It has good water solubility and good biocompatibility, and has good development and application prospects. According to the difference in molecular weight, γ-PGA can be used in many fields such as cosmetics, food, hygiene, medical treatment, and agriculture. [0003] There are three main ways to obtain γ-PGA: chemical synthesis, enzyme-catalyzed synthesis, and microbial fermentation. Among them, the chemical synthesis method and the enzyme catalysis method are difficult to be applied on a large scale due to the sma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00
CPCC12N13/00C12N15/01
Inventor 石元亮张雷王玲俐
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI