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High-throughput screening method for bacillus circulans

A technology of bacillus circulans and screening methods, which is applied in the fields of fermentation engineering and microbial breeding, can solve the problems of heavy screening workload and low screening efficiency, and achieve the effects of increasing screening capacity, simple screening methods, and increasing sample processing capacity

Inactive Publication Date: 2016-01-27
QUANTUM HI TECH (CHINA) BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Screening of mutant strains generally uses screening media to ferment and cultivate in shake flasks. This screening method has problems such as heavy screening workload and low screening efficiency.

Method used

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  • High-throughput screening method for bacillus circulans
  • High-throughput screening method for bacillus circulans
  • High-throughput screening method for bacillus circulans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Calculation method of enzyme activity:

[0039] 1. Reagent preparation

[0040] (1) Z-buffer

[0041] Dissolve 16.1g disodium hydrogen phosphate, 5.5g sodium dihydrogen phosphate, 0.75g potassium chloride, 0.246g magnesium sulfate and 2.7mL 2-mercaptoethanol in 800mL water, add 2mol / L sodium hydroxide solution, adjust the pH to 6.0± 0.05, detected with a pH meter. Transfer the solution to a 1000mL volumetric flask, dilute to volume with water, and mix well.

[0042] (2) ONPG (o-nitrophenyl-β-D-galactopyranoside) solution

[0043] Dissolve 250.0mg of ONPG with 75mL Z-buffer solution, transfer the solution to a 100mL volumetric flask, and use Z-buffer solution to make up volume as a substrate.

[0044] (3) Stop solution

[0045]Use water as the solvent, dissolve 10g of sodium carbonate, and transfer to a 100mL volumetric flask to constant volume.

[0046] (4) Test sample preparation

[0047] Test enzyme samples were prepared such that the final solution contained 0...

Embodiment 2

[0065] Follow the steps below for high-throughput screening of Bacillus circulans:

[0066] (1) starting bacterium activation: the bacillus circulans (concentration of spore is 10 5 ~10 8 individuals / mL) as the initial strain, streak culture, the initial strain was inoculated in 100mL fermentation medium after three times of slant LB plate activation, cultured in a shake flask at 37°C for 40h and then centrifuged, the supernatant was taken for β-half Lactosylase activity assay, its assay result is as shown in table 1:

[0067] Table 1 Initial strain β-galactosidase activity

[0068]

[0069] Pick a ring of slant strains in fresh LB medium, culture at 37°C for 6 hours, centrifuge at 10,000rmp for 10 minutes, collect the bacteria, resuspend them in sterile water, filter them through absorbent cotton, and place them in a small Erlenmeyer flask filled with glass beads , shake evenly, prepare a single-cell suspension, and adjust the cell concentration to 10 after counting wit...

Embodiment 3

[0078] Follow the steps below for high-throughput screening of Bacillus circulans:

[0079] (1) starting bacterium activation: the bacillus circulans (concentration of spore is 10 8 cells / mL) as the initial strain, carry out streak culture, pick a ring of slanted bacteria on a fresh LB plate, culture at 30°C for 12 hours, centrifuge at 10,000rmp for 10min, collect the bacteria, resuspend in sterile water, and pass through absorbent cotton After filtering, place it in a small Erlenmeyer flask equipped with glass beads, oscillate evenly, and prepare a single-cell suspension. After counting with an optical microscope, adjust the cell concentration to 10 6 About one / mL, spare;

[0080] (2) UV mutagenesis: place 5 mL of the cell suspension in step (1) at a distance of 25 cm from a 30w UV lamp, and irradiate for 30 min;

[0081] (3) Chlorine mutagenesis: after ultraviolet irradiation treatment, put the bacteria solution in ice bath for 2 hours, put it into fresh LB culture medium,...

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Abstract

The invention belongs to the technical field of fermentation engineering and microbial breeding, and discloses a high-throughput screening method for bacillus circulans for generating beta-galactosidase. The high-throughput screening method includes main steps of activating initial strains; carrying out ultraviolet mutation and lithium chloride mutation; carrying out high-throughput screening by the aid of 96 pore plates to obtain bacillus circulans mutant strains with improved enzyme capacity. The high-throughput screening method has the advantages that the mutation probability of the strains can be improved by the aid of the traditional physical and chemical combined mutation modes, the screening workload can be reduced by the aid of high-throughput screening tools, the screening efficiency can be improved, strain breeding procedures can be accelerated, and the optimal strains can be screened.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering and microbial breeding, and relates to a high-throughput screening method for bacillus circulans producing beta-galactosidase. Background technique [0002] Galactooligosaccharides are mainly produced from lactose by microbial β-D-galactosidase catalyzed galactosyl transfer reaction, and the products are mainly trisaccharides (4' or 6'-galactosyllactose) and a small amount of Four, five, six sugars. The chemical formula of galactooligosaccharide is (galactose) n-glucose, n=2-4. The types of glycosidic bonds connecting galactosyl and galactosyl in the structure of galactooligosaccharides are β(1→3), β(1→4), β(1→6), among which β(1→4) is the main . β-D-galactosidase can catalyze the transfer of galactosyl to the C2, C3, and C6 positions of glucose to generate galactosyl transfer disaccharides of all possible glycosidic bond types except β(1→1). [0003] The β-galactosidase produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12R1/09
Inventor 曾宪经邓宝浣陈振鹏阮鸿波陈子健
Owner QUANTUM HI TECH (CHINA) BIO CO LTD
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