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Acidophil Beta-glucanase GLU7A and gene and application thereof

A dextranase and gene technology, applied in the field of genetic engineering, can solve the problems of dextranase activity differences, achieve high enzyme activity stability, reduce material viscosity, and improve feed digestibility and metabolism.

Active Publication Date: 2013-04-03
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Genes from different sources can be expressed in different vectors and host systems, including Escherichia coli, Bacillus, Saccharomyces cerevisiae, and transgenic barley and tobacco, but the glucanase activity of different expression systems There is a big difference

Method used

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  • Acidophil Beta-glucanase GLU7A and gene and application thereof
  • Acidophil Beta-glucanase GLU7A and gene and application thereof
  • Acidophil Beta-glucanase GLU7A and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Acidophilic fungus Bispora sp.MEY-1 and its enzyme production characteristics

[0039] A sample of uranium mine wastewater from a mine in Jiangxi was cultured in potato juice medium, and then spread on enzyme-producing medium ((NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, CaCl 2 0.2g / L, 1% dextran, 1.5% agarose, pH 2.5) on a plate, cultured at 30°C for 5-6 days, a transparent circle can be seen on the enzyme-producing medium plate. Proved to have dextranase activity.

[0040] The acidophilic fungus Bispora sp.MEY-1 was preserved on May 19, 2008 in the General Microbiology Center of the China Committee for the Collection of Microorganisms (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101), and its preservation number For: CGMCC No.2500.

Embodiment 2

[0041] Example 2 Cloning of the glucanase-encoding gene glu7A from the acidophilic fungus Bispora sp.MEY-1

[0042] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:

[0043] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0044] According to the conserved sequence of g...

Embodiment 3

[0049] RT-PCR Analysis of Example 3 Glucanase Gene

[0050] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (glu7AF: 5′-ATGGCAGTGTTCACTCTGACCGTGG-3′, glu7AR: 5′-CTAATTCCAACCGCCAGAGCC-3′) to amplify The single-stranded cDNA obtains a cDNA sequence encoding glucanase, amplifies the product and recovers it for sequencing.

[0051] By comparing the genome sequence and cDNA sequence of glucanase glu7A, it was found that the gene has a 60bp intron, the cDNA is 1281bp in length, encoding 427 amino acids, and the first 19 amino acids at the N-terminal are possible signal peptide sequences. BLAST results showed that it had the highest sequence similarity with endo-1,4-beta-glucanase from Aspergillus oryzae. The highest amino acid similarity is 58%; the highest nucleotide similarity is 60.3%. It was proved that the gene encoding glucanase isolated and cloned from Bispora sp.MEY-1 was a new gene.

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Abstract

The invention relates to the field of genetic engineering, in particular to an acidophil Beta-glucanase GLU7A and gene and application thereof. The invention provides a glucanase GLU7A from acidophil bispora Bisporasp, and the amino acid sequence thereof is shown as SEQIDNO.1, and the invention provides a genome of coding the glucanase and cDNA coding gene glu7A. The invention obtains an acidophil glucanase which has the optimum pH value of 1.5-5.0, simultaneously remains high enzymatic activity in acid environment, and resists protease hydrolysis. In addition, the glucanase not only hydrolyzes Beta-1, 3-1, 4-glucanase, but also can hydrolyze cellulose. Due to the properties, the acidophil Beta-glucanase GLU7A can be applied in the industries of food, feedstuff and beer.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acidophilic beta-glucanase GLU7A and its gene, a recombinant vector containing the gene and application thereof. 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 bonds linking glucose to form cellotriose or Cellotetraose is connected to each other by β-1,3 bonds to form a linear mu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/46C12N15/56C12N15/63C12N1/21C12N1/19C12R1/19C12R1/85C12R1/07C12R1/225C12R1/645
CPCY02P20/52
Inventor 姚斌罗会颖王亚茹杨培龙孟昆史秀云柏映国袁铁铮石鹏君杨君
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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