Neutral low-temperature xylanase CaXyn10A, and gene and application thereof
A technology of xylanase and low temperature, applied in the field of genetic engineering
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Embodiment 1
[0047] Embodiment 1 Cladosporium acalyphae SL-16 produces enzyme characteristic
[0048] After culturing Cladosporium acalyphae SL-16 in potato juice medium, the spore suspension was made into bran liquid medium (Luo et al. Enzyme Microbial Technology, 2009, 45:126–133), 15 Cultivate at ℃ for 7 days, use 1% beech xylan as substrate, react at pH 6.0 and 30℃ for 10 minutes, and use DNS method (Miller 1959) to determine that it has xylanase activity.
Embodiment 2
[0049] Cloning of Example 2 Cladosporium acalyphae SL-16 Xylanase Encoding Gene Caxyn10A
[0050] Extraction of Cladosporium acalyphae SL-16 genomic DNA:
[0051] 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.
[0052] Design and synthesis of specific primers Caxyn10A-F / Caxyn10A-F:
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Embodiment 3
[0056] RT-PCR Analysis of Example 3 Cladosporium acalyphae SL-16 Xylanase Gene
[0057] Extract the total RNA of Cladosporium acalyphae SL-16, use reverse transcriptase to obtain a strand of cDNA, and then amplify the single-stranded cDNA with primers Caxyn10A-E-F / Caxyn10A-E-F to obtain the cDNA sequence of xylanase, After the amplified product was recovered, it was sent to Sanbo Biotechnology Co., Ltd. for sequencing.
[0058] Caxyn10A-E-F: 5'-ccccatatgatgctgatttccaacgccattgcc-3';
[0059] Caxyn10A-E-R: 5'-cccgaattcttagtggtggtggtggtggtgcaaagcgttgaggagagcagtg-3';
[0060] By comparing the genome sequence and cDNA sequence of xylanase, it is found that the gene has 4 introns, the cDNA is 999bp long, encodes 332 amino acids and a stop codon, and the N-terminal 18 amino acids are its signal peptide sequence. The measured partial nucleotide sequence of the mature protein of the gene Caxyn10A was homologously compared with the xylanase gene sequence on GenBank, and the isolated a...
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