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Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof

A technology of glucanase and recombinant strains, applied in the field of genetic engineering, can solve problems such as differences in glucanase activity

Active Publication Date: 2008-03-05
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Genes from different sources were expressed in different vectors and host systems, including Escherichia coli, Bacillus, Saccharomyces cerevisiae, and transgenic barley and tobacco, but the glucanase activities of different expression systems were different. big difference

Method used

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  • Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof
  • Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof
  • Beta-glucanase, encoding gene thereof, recombinant plasmid and bacterial strain and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Cloning of Paenibacillus β-glucanase Encoding Gene Bg1

[0059] Genomic DNA extraction of Paenibacillus spF-40: take Paenibacillus cultured at 30° C. for 2 days, and centrifuge at 10000 rpm for 10 min. Take 50 mg of bacteria and add 500 μL of sterile water to wash, and centrifuge to get the precipitate. Resuspend the precipitate in 500 μL lysozyme mixture, incubate at 37°C for 30 minutes, add 100 μL of enzyme solution and continue to incubate at 40-50°C for 30 minutes until the bacterial solution is transparent, add 10% SDS to a final concentration of 2%, and stir for about After 5 minutes until the viscosity of the bacterial liquid drops significantly, centrifuge at 15,000 rpm for 10 minutes to remove debris. The supernatant was extracted sequentially with equal volumes of phenol, phenol:chloroform, and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 16...

Embodiment 2

[0061] Example 2 Preparation of recombinant β-glucanase

[0062] After molecular biology software analysis, the sequence of the mature protein encoding β-glucanase was obtained, and the primer 1,5′-aaa was designed gaattcggttatgtgttctgggaacctc-3', (EcoR I site) and primer 2, 5'-acc gcggccgc ttaattactcgtatatttaaccc-3' (Not I site), the mature protein coding sequence was amplified by PCR. Digest with EcoR I and Not I, connect to vector pPIC9, obtain recombinant plasmid pPIC-Bgl containing Paenibacillus β-glucanase coding gene Bgl and transform Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain pPIC- Bgl / GS115.

[0063] Take the GS115 strain containing the recombinant plasmid, inoculate it in 200mL YPD culture medium, culture it overnight at 30°C with 250rpm shaking, and then inoculate it in a 3L fermenter based on basic fermentation. After a total of 156 hours in three stages of basal culture, carbon source feeding and induction culture, the recombinant β-glu...

Embodiment 3

[0064] Example 3 Purification of recombinant β-glucanase of the present invention

[0065] The fermentation culture liquid prepared in Example 2 was centrifuged at 10,000 rpm for 10 minutes to remove the bacteria, and the supernatant was taken as the crude enzyme liquid, and the crude enzyme liquid was placed in an ice bath, and ammonium sulfate was slowly added to 80% while stirring, and centrifuged at 13,000 rpm After 20 min, the precipitate was taken and redissolved with buffer to obtain a concentrated enzyme solution, which was further purified by HPLC (KTA FPLC, Pharmacia Company).

[0066] After being precipitated by ammonium sulfate, it was loaded on a HiTrap_Q_Sepharose_XL (Amersham Pharmacia Biotech prepacked column) anion column. Add 2 mL of sample, first elute the equilibrated column with pH 8.0, 0.02 mol / L Tris-HCl buffer solution, and then use the same buffer solution to prepare 0-0.6 mol / L NaCl gradient to elute 10 column beds (about 50 mL), The flow rate is 5m...

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Abstract

The present invention discloses one new kind of beta-glucanase, its coding gene, recombinant plasmid containing the coding gene and recombinant strain, and the preparation process and application in feed industry of the recombinant beta-glucanase. One new beta-glucanase gene is separated and cloned from bacillus Paenibacillus sp.F-40, and one expression plasmid capable of expressing the recombinant beta-glucanase in yeast cell is constituted. The beta-glucanase of the present invention has effect of decomposing beta-1, 3 bond and beta-1, 4 bond in beta-glucan, high specific activity, excellent heat stability and pH stability and other advantages, and may be applied in feed industry and beer industry.

Description

technical field [0001] The present invention relates to a glucanase, its encoding gene, in particular to a β-glucanase, its encoding gene, a recombinant plasmid and bacterial strain containing the gene, and a preparation method of the recombinant β-glucanase The invention and its application in the feed industry belong to the field of genetic engineering. Background technique [0002] β-glucan is a structural non-starch polysaccharide in the cell wall of higher plants of Poaceae, especially abundant in the endosperm cell wall of cereals (barley, oat, sorghum, rice and wheat). [0003] β-glucan is a polysaccharide polymer linked by D-type β-conformation glucose, which has a linear spatial structure. According to the different glycosidic linkages, β-glucan can be divided into 1,4-β-glucan, 1,3-β-glucan, 1,6-β-glucan, 1,3-1 , 6-β-glucan, 1,3-1,4-β-glucan, etc. Among them, 1,3-1,4-β-glucan is connected by mixed glycosidic bonds of β-1,3 and β-1,4, usually by β-1,4 glycosidic ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N9/42C12N15/56C12N15/63C12N1/19C12R1/84
Inventor 姚斌杨培龙王亚茹孟昆袁铁铮罗会颖柏映国史秀云
Owner WUHAN SUNHY BIOLOGICAL
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