Acidophil Beta-glucanase GLU7A and gene and application thereof
A glucanase and gene technology, applied in the field of genetic engineering, can solve problems such as differences in glucanase activity, achieve high enzyme activity stability, reduce environmental pollution, and increase productivity.
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Embodiment 1
[0038] Example 1 Acidophilic fungus Bispora sp. MEY-1 and its enzyme production characteristics
[0039] A sample of uranium ore wastewater from a mine in Jiangxi was cultured in potato juice medium, and then coated on an 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, dextran 1%, 1.5% agarose, pH 2.5) plate, cultured at 30°C for 5-6 days, and transparent circles can be seen on the enzyme production medium plate. Prove that it has glucanase activity.
[0040] The acidophilic fungus Bispora sp.MEY-1 was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101) on May 19, 2008, and its collection number It is: CGMCC No.2500.
Embodiment 2
[0041] Example 2 Cloning of glu7A encoding gene glu7A from the acidophilic fungus Bispora sp.MEY-1
[0042] Extract genomic DNA from the acidophilic fungus Bispora sp.MEY-1:
[0043] Filter the mycelium cultured for 3 days with sterile filter paper and put it in a mortar, add 2 mL of extract, grind for 5 min, then place the grind in a 50 mL centrifuge tube, lyse in a water bath at 65°C for 20 min, and mix every 10 min. Homogenize once and centrifuge at 10,000 rpm at 4°C for 5 min. The supernatant was extracted in phenol / chloroform to remove impurities, and then the supernatant was added to an equal volume of isopropanol. After standing at room temperature for 5 min, centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved in an appropriate amount of TE, and placed at -20°C for use.
[0044] Degenerate primers P1: 5'-ggYTACTgYgAYgCNCARTg-3'; and P2: 5'-TAgggRTTgAANCCRCANCC-3' were design...
Embodiment 3
[0049] Example 3 RT-PCR analysis of 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 From the single-stranded cDNA, a cDNA sequence encoding a glucanase is obtained, and the amplified product is recovered and sequenced.
[0051] By comparing the genomic sequence and cDNA sequence of glucanase glu7A, it is found that the gene has a 60bp intron. The full length of the cDNA is 1281bp, which encodes 427 amino acids. The first 19 amino acids at the N-terminal are possible signal peptide sequences. The BLAST alignment results showed that it had the highest sequence similarity with endo-1,4-beta-glucanase derived from Aspergillus oryzae. The highest similarity of amino acids is 58%; the highest similarity of nucleotides is 60.3%. It proved that the gene encoding glucanase isolated and cloned ...
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