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Xylanase XYNA4 with wide pH applicability and gene and application thereof

A xylanase and gene technology, applied in the field of genetic engineering, can solve the problems of cloning and expression without related reports

Active Publication Date: 2010-05-05
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

At present, there is no relevant report on the xylanase gene derived from Alicyclobacillus and its cloning and expression

Method used

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  • Xylanase XYNA4 with wide pH applicability and gene and application thereof
  • Xylanase XYNA4 with wide pH applicability and gene and application thereof
  • Xylanase XYNA4 with wide pH applicability and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Isolation and purification of Alicyclobacillus hesperidum A4 (CGMCC No.3147) and its enzyme-producing properties

[0047] Soil samples collected from high hot springs in Yunnan were transferred to enrichment medium (yeast extract 1.0g, tryptone 2.0g adjusted to pH 2.0 with hydrochloric acid), cultured at 60°C for 72 hours, diluted and plated to the separation medium ( Yeast extract 1.0g, tryptone 2.0g, oat xylan 5.0g, agar 30, Congo red 0.5g, adjust the pH to 3.0 with hydrochloric acid) After culturing at 60°C for 72 hours, pick according to the presence and size of the transparent circle An enzyme-producing strain, numbered A4, was identified as Alicyclobacillus hesperidum A4 based on 16SRNA and strain characteristics. Alicyclobacillus hesperidum A4 was then transferred to the growth medium (1.0 g of yeast extract, 2.0 g of tryptone and adjusted to pH 3.0 with hydrochloric acid), and its optimum growth temperature and pH value were tested. The results showed...

Embodiment 2

[0048] Example 2 Cloning of Alicyclobacillus hesperidum A4 (CGMCC No.3147) Xylanase Encoding Gene XYNA4

[0049] Extract Alicyclobacillus hesperidum A4 (CGMCC No.3147) genomic DNA:

[0050] Centrifuge the cultured bacteria for 2 days, add 1mL lysozyme, treat at 37°C for 60min, then add lysate, lyse in a water bath at 65°C for 30min, mix every 10min, and centrifuge at 10,000rpm at 4°C for 5min. 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.

[0051] The degenerate primers XYN10F and XYN10R were designed and synthesized according to the conserved (YDWDV and HGIGM) sequences of the tenth family xylanas...

Embodiment 3

[0058] Example 3 Preparation of recombinant xylanase.

[0059] The expression vector PET-22B(+) was double digested (EcoRI+Hind III), and the gene XYNA4 encoding xylanase was double digested (EcoRI+Hind III) to cut out the gene encoding mature xylanase The fragment was connected with the expression vector pPET-22B(+), and the recombinant plasmid PET-22b(+)-XYNA4 containing Alicyclobacillus hesperidum A4 (CGMCC No.3147) xylanase gene XYNA4 was obtained and transformed into Escherichia coli BL21(DE3), and obtained Recombinant E. coli strain BL21(DE3) / XYNA4.

[0060] Take the BL21(DE3) strain containing the recombinant plasmid, inoculate it in 200mL LB culture medium (1000ml Erlenmeyer flask), shake it at 250rpm at 37°C for 3-4h, and add IPTG for induction. After shaking culture at 250 rpm at 30°C for 10-12 hours, the supernatant was collected by centrifugation. Determination of xylanase activity. The expression level of recombinant xylanase was 0.38U / mL. After the expressed ...

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Abstract

The invention relates to the gene engineering field, in particular to a xylanase XYNA4 and gene and application thereof. The invention provides a xylanase XYNA4 derived from alicyclic acid bacillus Alicyclobacillus hesperidumA4 (CGMCC No. 3147), amino acid sequence thereof is shown as SEQ ID NO. 1, and the invention also provides gene xynA4 coding the xylanase. The xylanase of the invention has the following properties: optimal pH is 7.0, optimal temperature is 55 DEG C, and specific activity is 4.20.2U / mg; and the xylanase has pH stability with extreme range, higher pH adaptability and better thermostability. The xylanase is used as a novel enzymic preparation can be widely applied to industries of animal feed, food, papermaking and energy sources.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a wide pH adaptive xylanase XYNA4 and its gene and application. Background technique [0002] Xylan (xylan) is the main component of plant hemicellulose, widely exists in nature, and its content is second only to cellulose as the second most abundant polysaccharide (Collins et al. FEMS Microbiology Reviews. 2005, 29: 3 -twenty three.). Xylanase is a general term for a class of enzymes that can degrade xylan into oligosaccharides and xylose. At present, xylanase has been widely used in the fields of feed, bread, beer, paper, textile, medicine and energy. In the paper industry, xylanase can be applied to biopulping, pulp bleaching, waste paper deinking treatment, etc., especially in pulp biobleaching, xylanase cooperates with traditional chlorine bleaching to promote The degradation of residual lignin and the extraction of soluble lignin can not only imp...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12N1/20A23K1/165A21D8/04C12C7/00C12P19/14D21C9/10C12R1/19C12R1/07C12R1/225C12R1/865C12R1/84C12R1/78C12R1/01A23K20/189
Inventor 姚斌柏映国石鹏君罗会颖黄火清杨培龙孟昆赵珩王亚茹史秀云袁铁铮
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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