Acidic xylanase XYL10A and gene and application thereof
A technology of acid xylanase and xylanase, applied in the field of genetic engineering
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Embodiment 1
[0122] Example 1 Isolation of acidophilic fungus Bispora sp.MEY-1
[0123] After the uranium mine wastewater sample from a mine in Jiangxi was enriched and cultured (enrichment 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, 0.5% corncob powder, 0.5% bran, pH2.5), and spread it on the 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% xylan, 1.5% agarose, pH 2.5) on a plate, culture at 30°C for 5-6 days, pick colonies that produce transparent circles, and streak and separate on the enzyme-producing medium plate, repeat streaking and separation Process 3 rounds to purify the strain. The strain that secretes xylanase is screened by this method.
[0124] This strain was cultured on PDA at 30°C for 7 days, and the colony diameter was 2-3cm, gray-black or gray-brown, circular and radial, with velvet-like wrinkles on the sur...
Embodiment 2
[0125] Example 2 Cloning of the gene xyl10A encoding the xylanase of the acidophilic fungus Bispora sp.MEY-1
[0126] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:
[0127] 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, 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.
[0128] Degenerate primers P1, P2 were designed an...
Embodiment 3
[0136] RT-PCR analysis of embodiment 3 xylanase gene
[0137] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (Xyl10AF: 5′-ATGTCTTTTCCACTCGCTTCTAATCTCAGGTCTTC-3′, Xyl10AR: 5′-TCATGGACTTTCCGCCTTATGTTGCAAAGCC-3′) to amplify The single-stranded cDNA is obtained from the cDNA sequence of xylanase, and the amplified product is recovered and then sent to Sanbo Biotechnology Co., Ltd. for sequencing.
[0138] By comparing the genome sequence and cDNA sequence of xylanase, it was found that the gene has an intron, the cDNA is 1275bp long, encodes 424 amino acids and a stop codon, and the N-terminal 18 amino acids are its predicted signal peptide sequence. The measured nucleotide sequence of the mature protein part of the gene xyl10A was homologously compared with the xylanase gene sequence on GeneBank, and the highest identity was 57.1%, and the highest amino acid sequence identity was 45.7%, which pr...
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