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Rapid high-flux screening technology of beta-glucosidase producing strain

A glucosidase, high-throughput technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low efficiency, high cost, time-consuming, etc., and achieve a simple screening method, less reagent consumption, low cost effect

Inactive Publication Date: 2012-03-28
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the problems of time-consuming, laborious, low efficiency and high cost in the current method for screening β-glucosidase-producing bacteria, the present invention provides a high-throughput screening method for β-glucosidase-producing bacteria based on a 96-well plate, In order to effectively solve the above-mentioned problems, it is only applicable to the rapid and high-throughput primary screening of β-glucosidase-producing bacteria, and further verification is required for the secondary screening

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Rapid screening of active clones producing β-glucosidase from the bovine feces microbial plasmid metagenomic library

[0023] Samples of the plasmid metagenomic library (containing 8,115 single clones) of Miscanthus-fed cow fecal microorganisms were stored in a 96-well plate in a -80°C freezer. Prepare 100 mL of LB medium containing Ampicillin (100 mg / mL) and sterilize it, randomly take some samples from its library and revive 370 single colonies, add 100 μL of Ampicillin (Ampicillin) to each 96-well plate (100 mg / mL) LB medium and 5 μL of preserved bacterial liquid, cultivated at 37 ° C, 120-150 rpm for 10-12 h; the revived bacterial liquid was expanded, and 93 μL of Ampicillin (100 mg / mL) was added to each microwell of a 96-well plate. mL) LB medium and 4 μL of the bacterial solution of the bacteria to be screened, cultured at 37°C, 120-150 rpm for 6-8 hours; for a display reaction, add 3 μL of 10mM pNPG to each microwell of a 96-well plate, and set a negat...

comparative example 1

[0024] Comparative Example 1: Rapid screening of active clones producing β-glucosidase from bovine feces microbial plasmid metagenomic library using aescin plate method

[0025] Prepare 40 mL of LB medium containing Ampicillin (100 mg / mL) and sterilize it, randomly take some samples from its library and revive 370 single colonies, add 100 μL of Ampicillin (Ampicillin) to each 96-well plate (100mg / mL) LB medium and 5μL preserved bacteria solution, cultured at 37°C, 120-150rpm for 10-12h; prepared 3700mL escin medium and sterilized to make 370 culture plates; each took 5μL revived bacteria solution and directly spot On the plate containing escin medium, after inverting at 37°C for 24-48 hours, observe the 370 culture plates that have been inoculated one by one. According to the black hydrolysis circle around the colony, it was preliminarily judged whether the strain produced β-glucosidase. Results A escin plate method was used to screen the library and one positive clone (M16) ...

Embodiment 2

[0026] Example 2: Rapid screening of β-glucosidase-producing functional strains in termite intestinal microorganisms

[0027] Termites were collected in the dead wood forest of Hunan Provincial Botanical Garden, and the intestinal microorganisms of termites were taken in the ultra-clean workbench and dissolved in sterile water, fully shaken and mixed, and then gradiently diluted 10 5 , 10 6 times, and then pipette 0.1mL of the diluted solution onto the LA plate, and culture it at 37°C for 12-24h; 243 strains of bacteria were isolated.

[0028] Prepare 300mL LB medium and sterilize it, inoculate 243 strains of bacteria into 1mL LB liquid medium respectively, culture at 37°C, 180-200rpm for 10-12h for resurrection; Add 93 μL of LB medium and 4 μL of revived bacteria solution, and incubate for 8-12 hours at 37°C, 120-150 rpm; add 3 μL of 10 mM reaction substrate pNPG to a 96-well plate, and react for 40 minutes at 37°C, 120-150 rpm, and set up negative controls and positive con...

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PUM

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Abstract

The invention provides a rapid high-flux screening technology of beta-glucosidase producing strain based on a 96-orifice plate, and especially a method for adopting pNPG in high-flux screening of beta-glucosidase. The method comprises specific steps of: (1) strain reactivation; (2) reacted bacterial liquid amplification; (3) color reaction; (4) absorbance determination, and the like. According to the invention, beta-glucosidase producing microbe strains can be mined by using a pure culture isolation technology and a gene library establishing technology. Compared to a currently reported esculin plate method and a fluorometric method used in beta-glucosidase screening, the method for adopting pNPG in beta-glucosidase screening provided by the invention has advantages of convenient operation, high speed, accurate result, low price, and the like.

Description

technical field [0001] The invention relates to a method for high-throughput screening of β-glucosidase-producing bacteria based on a 96-well plate. Background technique [0002] Cellulose is an earth-abundant and renewable biopolymer. Due to its complex structure, the cellulose degradation process depends on the synergistic action of a complex multi-enzyme system, namely endo-cellulase, exo-cellulase and β-glucosidase, among which β-glucosidase is The rate-limiting enzyme for this process. Pure culture isolation technology and gene library construction technology are the main strategies for mining microbial resources in environmental samples. At present, the main screening method for β-glucosidase-producing bacteria is based on functional screening. It has been reported in the literature that the research strategy of using functional screening to mine β-glucosidase-producing bacteria is based on the fluorescence method and the escin chromogenic substrate plate method. Us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34C12Q1/04
Inventor 田云卢向阳刘虎虎赵飞辛盛
Owner HUNAN AGRICULTURAL UNIV
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