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 limited application and high production cost

Active Publication Date: 2010-09-22
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

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 Enzyme production characteristics of Penicillium sp. F63 CGMCC1669

[0056] The Penicillium sp.F63 CGMCC1669 was cultured in potato juice medium and then spread on the 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, pH 5.0) plate, cultured at 30°C for 5-6 days, transparent circles can be seen on the plate with enzyme production medium. Prove that it has xylanase activity.

Embodiment 2

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

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

[0059] 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.

[0060] Degenerate primers P1, P2 were designed and synthesized according to the conservative (CYLG(A)VYGW and TFNQYWS) ...

Embodiment 3

[0069] Example 3 RT-PCR analysis of xylanase gene

[0070] Extract the total RNA of Penicillium sp.F63 (CGMCC166), use reverse transcriptase to obtain a strand of cDNA, and then design the appropriate primers (Xy110A F: 5'-ATGGTCTCTTTTTCAAACCTCTTTATGGCTGCCTG-3', Xy110A R: 5'-TTAGGAAACAGTGATGGACGAAGAGCCACT-3' ) 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 of xylanase with the cDNA sequence, it was found that the gene has an intron. The cDNA is 654 bp in length, encoding 217 amino acids and a stop codon, and the N-terminal 19 amino acids are its predicted signal peptide sequence. The detected partial nucleotide sequence of the mature protein of the gene xyn11F63 was compared with the xylanase gene sequence on GeneBank. The highest identity was 86%, and the highest identity of the amino acid sequence was 85%, which prov...

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PUM

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Abstract

The 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 genes xyn11F63. 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 present invention relates to the field of genetic engineering. Specifically, the present invention relates to a high specific activity xylanase XYN11F63 and its gene, a recombinant vector containing the gene, and applications. Background technique [0002] Xylan (xylan) is the main component of plant cell wall hemicellulose. It is the second most abundant polysaccharide in nature after cellulose, and it accounts for almost one-third of the earth's renewable organic carbon content (Collins et al. FEMS Microbiology Reviews. 2005, 29: 3-23.). For a long time, agricultural by-products rich in xylan, such as corn cobs, straws, bagasse, etc., are difficult to effectively use. At the same time, industrial wastes rich in xylan and chloride in the paper pulping process are often directly discharged into water bodies, causing water pollution such as eutrophication and severe damage to the ecological environment (Polizeli et al. Applied Microbiology and Biotechno...

Claims

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Application Information

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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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