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High-specific-activity xylanase XYN11F63 and genes and application thereof

A technology of XYN11F63 and xyn11f63, applied in the field of genetic engineering, can solve the problems of application limitations and high production costs

Active Publication Date: 2011-09-07
JIANGSU YINONG BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the application of xylanase derived from fungi is limited due to the high production cost

Method used

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  • High-specific-activity xylanase XYN11F63 and genes and application thereof
  • High-specific-activity xylanase XYN11F63 and genes and application thereof
  • High-specific-activity xylanase XYN11F63 and genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Penicillium sp.F63 CGMCC1669 enzyme production characteristics

[0056] After Penicillium sp.F63CGMCC1669 was cultured in potato juice medium, it was spread on enzyme production 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 O0.01g / L, CaCl 2 0.2g / L, 1% xylan, 1.5% agarose, pH5.0) plate, cultured at 30°C for 5-6 days, a transparent circle can be seen on the enzyme-producing medium plate. Prove that it has xylanase activity.

Embodiment 2

[0057] Example 2 Cloning of Penicillium sp.F63 (CGMCC1669) xylanase coding gene xyn11F63

[0058] Extract Genomic DNA of Penicillium sp.F63 (CGMCC1669):

[0059] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL 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.

[0060] The degenerate primers P1 and P2 were designed and synthesized according t...

Embodiment 3

[0069] The RT-PCR analysis of embodiment 3 xylanase gene

[0070] Extract the total RNA of Penicillium sp.F63 (CGMCC166), use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (Xyl10A F: 5′-ATGGTCTCTTTTTCAAACCTCTTTATGGCTGCCTG-3′, Xyl10A R: 5′-TTAGGAAACAGTGATGGACGAAGAGCCACT-3′ ) to amplify the single-stranded cDNA to obtain the cDNA sequence of xylanase, and the amplified product is recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0071] By comparing the genome sequence and cDNA sequence of xylanase, it was found that the gene has an intron, the cDNA is 654bp long, encodes 217 amino acids and a stop codon, and the N-terminal 19 amino acids are its predicted signal peptide sequence. The measured nucleotide sequence of the mature protein part of the gene xyn11F63 was homologously compared with the xylanase gene sequence on GeneBank, and the highest consistency was 86%, and the highest amino acid sequence consistency was 85%...

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Abstract

The present invention relates to the field of genetic engineering, in particular to high-specific-activity xylanase XYN11F63 and genes and application thereof. The invention provides xylanase XYN11F63 derived from Penicillium sp.F63 (CGMCC No.1669), wherein the amino acid sequences thereof is shown in SEQ ID No.1; and the invention further provides the genome for coding the xylanase and cDNA-coding genesxyn11F63. The xylanase of the invention has the following properties: the optimal pH value thereof is 4.5, the optimal temperature thereof is 40 DEG C, and the specific activity thereof is 7,988U / mg, and the xylanase has good proteinase resistance and is suitable for industrialized fermentation production; and the xylanase is widely applicable as a novel enzymic preparation in the industries of animal feed, food, papermaking, energy and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high specific activity xylanase XYN11F63 and its gene, a recombinant vector containing the gene and application thereof. Background technique [0002] Xylan (xylan) is the main component of plant cell wall hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, accounting for almost one-third of the renewable organic carbon content of the earth (Collins et al. FEMS Microbiology Reviews. 2005, 29: 3-23.). For a long time, xylan-rich agricultural by-products such as corncobs, straw, bagasse, etc. have been difficult to be effectively utilized. At the same time, industrial wastes rich in xylan and chloride are often directly discharged into the water body during the pulping process, leading to water pollution such as eutrophication and serious damage to the ecological environment (Polizeli et al.Applied Microbiology and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19A23K1/165C12P19/14C12P19/02C12R1/80C12R1/225C12R1/07C12R1/19
Inventor 项有炜石鹏君王娟娟张慧君
Owner JIANGSU YINONG BIOLOGY CO LTD
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