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A screening method for producing chitosanase strain

A technology for producing chitosanase and a screening method, applied in the field of microorganisms, can solve the problems of uneven distribution of chitosan, affecting the accuracy of screening results, inability to efficiently screen high-yielding acid chitosanase strains, etc. The effect of improved technology

Inactive Publication Date: 2017-10-24
GRAPE & WINE RES INST GUANGXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because the culture medium used must have pH ≥ 5.0, there are some defects in the existing screening methods: first, chitosan is in a water-insoluble precipitation state in an environment with pH ≥ 5.0, which can easily cause the distribution of chitosan in the medium. Inhomogeneity, which affects the accuracy of screening results
Therefore, in industrial applications, there is a greater need for acidic chitosanases with high activity and high stability under acidic conditions of pH<5.0, and the existing screening methods cannot realize the presentation of chitosanases on the screening plate under the conditions of pH<5.0. The reaction of polycanase to hydrolyze soluble colloidal chitosan, so it is impossible to efficiently screen for strains with high acid chitosanase production

Method used

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  • A screening method for producing chitosanase strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 produces the screening method of chitosanase

[0022] 1) Preparation of screening plates:

[0023] A. Preparation of colloidal chitosan: Use commercially available chitosan powder with a deacetylation degree of more than 75%, add 12mol / L concentrated hydrochloric acid at a mass-volume ratio of 5:3, fully stir for acidification, add water to dissolve the chitosan concentration It is 10g / L, promptly obtains the colloidal chitosan of corresponding concentration;

[0024] B. Preparation of screening plate culture medium: select ordinary beef extract-peptone medium (screening bacteria), replace the carbon source (glucose) in the medium with colloidal chitosan, and the addition amount of colloidal chitosan is Add 1g to liter medium;

[0025] C. Preparation of the screening plate: After the prepared screening plate medium was sterilized by damp heat at 121° C. for 20 minutes, the screening plate was made by inverting the plate according to the plate inversion me...

Embodiment 2

[0029] Embodiment 2 produces the screening method of chitosanase

[0030] 1) Preparation of screening plates:

[0031] A. Preparation of colloidal chitosan: use commercially available chitosan powder with a deacetylation degree of more than 75%, add glacial acetic acid at a mass-volume ratio of 5:1, fully stir for acidification, add water to dissolve in chitosan at a concentration of 100g / L, promptly obtain the colloidal chitosan of corresponding concentration;

[0032] B. Preparation of screening plate medium: select Chase medium (screening mold), replace the carbon source material (glucose) with colloidal chitosan, and the addition amount is 20g / L;

[0033] C. Preparation of the screening plate: After the prepared screening plate medium was sterilized by damp heat at 121° C. for 20 minutes, the screening plate was made by inverting the plate according to the plate inversion method in the basic operation of microbiology.

[0034] 2) Cultivation of single colony: According ...

Embodiment 3

[0037] Embodiment 3 produces the screening method of chitosanase

[0038] 1) Preparation of screening plates:

[0039] A. Preparation of colloidal chitosan: use commercially available chitosan powder with a deacetylation degree of more than 75%, add 12mol / L hydrochloric acid at a mass-volume ratio of 5:2, fully stir and acidify, add water to dissolve in chitosan with a concentration of 80g / L, promptly obtain the colloidal chitosan of corresponding concentration;

[0040] B. Preparation of screening plate medium: select Gaoshi No. 1 medium, replace the carbon source (soluble starch) with colloidal chitosan, and the addition amount is 5g / L;

[0041] C. Preparation of the screening plate: After the prepared screening plate medium was sterilized by damp heat at 121° C. for 20 minutes, the screening plate was made by inverting the plate according to the plate inversion method in the basic operation of microbiology.

[0042] 2) Cultivation of single colony: According to the separa...

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Abstract

The invention provides a screening method of a chitosanase-producing strain, which comprises the following steps: 1) preparation of screening plate; 2) culture of single colony; 3) plate dyeing; and 4) strain screening. By using the iodine sublimation dyeing process, the chitosan hydrolysis reaction by chitosanase (appearance of a transparent ring) can be quickly detected by a plate culture process under acidic conditions (the pH value is lower than or equal to 5.0), thereby screening out the high-chitosanase-yield strain according to the size of the transparent ring; and the screened strain can grow well and efficiently produce chitosanase under acidic conditions. The screening method provides technical assurance for screening excellent acidic chitosanase from the natural world and generating a new strain, is applicable to breeding and yield increase of the existing acidic chitosanase industrial production strain, and has important meanings for enhancing the technical level of industrial production of chitosanase.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a screening method for chitosanase-producing strains. Background technique [0002] Chitosanase is a key enzyme that catalyzes the hydrolysis of chitosan to produce chitooligosaccharides and glucosamine, and has broad application value in the fields of biopesticides, biopharmaceuticals, and fine chemicals. The source of existing chitosanase is mainly produced by fermentation of microbial strains. Therefore, how to effectively and quickly screen or select high-yielding microbial strains of chitosanase is very important for the industrial production of chitosanase. At present, researchers use the following method for the screening of chitosanase: common beef extract-peptone medium (screening bacteria), Gaoshi No. 1 medium (screening actinomycetes), Chase medium (screening mold) And other carbon source substances (such as sugars) in the microbial culture medium are replaced by colloid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/00C12Q1/04
CPCC12Q1/04G01N2333/942
Inventor 李玮张瑛周咏梅韦荣福时晓芳林玲曹雄军
Owner GRAPE & WINE RES INST GUANGXI ACADEMY OF AGRI SCI