A screening method for β-mannanase engineering bacteria

A mannanase and screening method technology is applied in the field of in situ rapid semi-quantitative screening of beta-mannanase mutants, which can solve the problem of inability to efficiently realize large-scale screening of mutant libraries, cumbersome cells, and energy consumption. wall treatment and other issues, to achieve the effect of facilitating high-throughput screening, rapid detection, and simple steps

Inactive Publication Date: 2021-07-20
SOUTH CHINA UNIV OF TECH
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Problems solved by technology

However, since the recombinant β-mannanase is mostly expressed intracellularly, and the substrate mannan is a macromolecular compound, it cannot freely enter and exit the cell wall of E. coli. Broken wall treatment
In particular, when random mutations are introduced into β-mannanase, the mutation pool usually reaches 10 3 For the order of magnitude above, cell wall breaking, recombinase purification, and enzyme activity detection are carried out for each mutant one by one. The detection cycle, manpower and material resources are very huge, and it is impossible to efficiently realize large-scale screening of mutant libraries.

Method used

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  • A screening method for β-mannanase engineering bacteria
  • A screening method for β-mannanase engineering bacteria
  • A screening method for β-mannanase engineering bacteria

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Embodiment 1

[0026] 1. Preliminary in situ verification of recombinant β-mannanase activity

[0027] First prepare the KT plate for screening. Prepare 1% trypan blue solution, filter and sterilize for later use. Prepare a solution containing 1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar and 0.5% konjac gum, and add a certain amount of 1% trypan blue solution to a final concentration of 0.03%. Spread a 90mm plate for every 20mL medium. Trypan blue and konjac gum can be closely combined, and the prepared flat plate is uniform blue.

[0028] Use a sterile toothpick to pick the β-mannanase expression strains E.coli BL21(DE3) / pET30-man25 and E.coli BL21(DE3) preserved in our laboratory onto the newly prepared KT plate. Wherein, E.coli BL21 (DE3) was used as a control group, and the β-mannanase expression strain E.coli BL21(DE3) / pET30-man25 was to insert the β-mannanase encoding gene into the pET30a expression vector Nde I and It was constructed in the Xho I restriction site...

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Abstract

The invention discloses a screening method for β-mannanase engineering bacteria, comprising the following steps: (1) cultivating the expression bacteria of β-mannanase on a KT plate containing trypan blue until the β-mannanase is produced on the plate (2) measure the diameter of the hydrolysis circle and the diameter of the microbial colony, and use the ratio of the two sizes as the enzyme activity index of β-mannanase, which is positively correlated with the enzyme activity. The invention can realize rapid detection of intracellular β-mannanase enzyme activity without lysing engineering bacteria. The required steps are simple, time-consuming, low-cost, and conducive to high-throughput screening.

Description

technical field [0001] The invention relates to an in-situ rapid semi-quantitative screening method for beta-mannanase mutants, which belongs to the field of bioengineering. Background technique [0002] β-mannanase (β-mannanase, EC 3.2.1.78) belongs to hemicellulase, which can randomly cut β-1,4-D- Mannosidic linkages, resulting in mannose oligosaccharides of varying lengths. As an additive, mannose oligosaccharides are widely used in the animal feed industry and are also an important component of dietary fiber. In addition, they also have important application values ​​in the fields of papermaking, textile industry, medicine, pharmacy, food and oil exploration. [0003] The expression of β-mannanase in Escherichia coli has the advantages of high expression, short culture period and low cost. However, since the recombinant β-mannanase is mostly expressed intracellularly, and the substrate mannan is a macromolecular compound, it cannot freely enter and exit the cell wall o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12Q1/34C12R1/19
CPCC12N1/20C12N9/2494C12Q1/34C12Y302/01078G01N2333/924
Inventor 李爽樊吴迪
Owner SOUTH CHINA UNIV OF TECH
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