Neutral low-temperature xylanase CaXyn10A, and gene and application thereof

A technology of xylanase and low temperature, applied in the field of genetic engineering

Active Publication Date: 2016-09-21
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

So far, only one low-temperature xylanase line has been reported (Del-Cid A et al. Applied Biochemistry and

Method used

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  • Neutral low-temperature xylanase CaXyn10A, and gene and application thereof
  • Neutral low-temperature xylanase CaXyn10A, and gene and application thereof
  • Neutral low-temperature xylanase CaXyn10A, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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:

[0...

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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Abstract

The invention relates to the field of genetic engineering, in particular to a neutral low-temperature xylanase CaXyn10A, and a gene and application thereof. The amino acid sequence of the neutral low-temperature xylanase CaXyn10A is shown as SEQ ID NO. 1. The xylanase is characterized in that the optimal pH is 6.0-6.5, the optimal temperature is 40 DEG C, over 20% of enzyme activity is kept at the temperature of 0 DEG C, and the specific activity is 453U/mg; the xylanase has activities of xylanase, glucanase and cellulose; industrialized fermentation production is facilitated. The neutral low-temperature xylanase CaXyn10A serving as a novel broad-spectrum preparation can be widely applied to aquatic feed, food, papermaking, energy industry and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a neutral low-temperature xylanase CaXyn10A and its gene and application. Background technique [0002] Xylan (xylan) is the main component of plant biomass, its content in nature is second only to cellulose (Lyndet al. Microbiology and Molecular Biology Review 2002,66:506–577), and has potential use in bioenergy value. Existing biotechnology can efficiently degrade the cellulose component in plants and convert it into fermentable glucose, which is produced by yeast to produce bioethanol. High-component xylan not only limits the combination reaction of cellulase and cellulose, inhibits the catalytic reaction, but also discharges into nature as a by-product, which can cause nutrient enrichment and damage the ecosystem (Polizeli et al. Applied Microbiology and Biotechnology. 2005, 67:577-591). Therefore, in the process of biomass bioconversion, xylanase ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63
CPCC12N9/248
Inventor 姚斌马锐柏映国黄火清罗会颖苏小运王亚茹王苑师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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