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Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof

A xylanase, cold-adapting technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of difficulty in sampling and cultivation, less research on polar microorganisms, etc., to ensure safety, high enzyme activity, and energy saving. Effect

Inactive Publication Date: 2012-07-04
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to difficulties in sampling and cultivation, there are relatively few studies on polar microorganisms.

Method used

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  • Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof
  • Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof
  • Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A marine cold-adapted endo-beta-xylanase XynB, the amino acid sequence of which is shown in SEQ ID NO.1. The nucleotide sequence of the gene xynB encoding the marine cold-adapted endo-β-xylanase XynB is shown in SEQ ID NO.2.

[0032] The gene xynB is 2739bp in total, which contains a 2739bp open reading frame encoding the cold-adapted endoxylanase XynB, the start codon is located at 1bp, the stop codon is located at 2737bp, and a total of 912 amino acids are encoded.

Embodiment 2

[0033] Embodiment 2: Cloning method of gene xynB

[0034] The strain source: Glaciecola mesophila KMM241 was purchased from the German Culture Collection of Microorganisms (DSM), and the strain preservation number is 15026.

[0035] Specific steps are as follows:

[0036] 1. Cloning of gene encoding cold-adapted endoxylanase XynB

[0037] 1.1 The extraction of Glaciecola mesophila KMM241 genomic DNA refers to the "Refined Molecular Biology Experiment Guide":

[0038] (1) Cultivate 50ml of bacterial culture to saturation, take 15ml of culture and centrifuge at 12000g for 5min;

[0039] (2) Add 5670 μl TE to the precipitate, resuspend it by pipetting repeatedly with a pipette, then add 300 μl 10% SDS and 30 μl 20mg / mL proteinase K, mix well, and incubate at 37°C for 2.5-3h;

[0040] (3) Add 1000 μl 5M NaCl, mix well, then add 800 μl CTAB / NaCl solution, mix well, and incubate at 65°C for 30-60min;

[0041] (4) Add an equal volume of mixed solution (the mixed solution is prepare...

Embodiment 3

[0069] Example 3: Heterologous expression and purification of xylanase XynB

[0070] 3. Expression of gene xynB in Escherichia coli

[0071] 3.1 Construction of expression vector pet-22b-xynB

[0072] (1) Using a small-dose plasmid extraction kit, follow the steps in the instructions to extract the recombinant plasmid pGEMT-xynB from the transformant containing the recombinant pGEMT plasmid.

[0073] (2) Design two specific primers according to the sequencing results

[0074] XYB1: CGAC GGATCC GAGCACTTTTATCAGTCAG (SEQ ID NO. 5) is underlined as the BamHI restriction site. XYB2: CGACG CTCGAG TTCAGGCTCGTTTTC (SEQ ID NO. 6) underlined is the XhoI restriction site. The above primers were synthesized by Shanghai Sangon Biotechnology Company.

[0075] (3) PCR is used to amplify the gene sequence, using XYB1 and XYB2 as primers, and using the recombinant pGEMT plasmid as a template for PCR amplification. The PCR reaction conditions were: 95° C. for 5 minutes; then 95° C. for...

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Abstract

The invention relates to a marine cryophilic endo beta-xylanase XynB as well as an expressing gene xynB and an application thereof, belonging to the technical field of biotechnology. The invention provides a marine cryophilic endo beta-xylanase XynB the amino acid sequence of which is shown in the SEQ ID NO.1 and gene xynB for coding the marine cryophilic endo beta-xylanase XynB the nucleotide sequence of which is shown in the SEQ ID NO.2. The beta- xylanase XynB provided by the invention has the characteristic of high catalytic efficiency at 0-35 DEG C,can be applied to pulp bleaching, xylooligosaccharide production and flour modification at room temperature and ensures that the step of warming up in the traditional process is omitted so as to save energy.

Description

technical field [0001] The invention relates to a marine cold-adapted endo-beta-xylanase and its expression gene xynB and its application, belonging to the technical field of biotechnology. Background technique [0002] β-xylanase is a glycoside hydrolase that is secreted by bacteria and fungi and can degrade xylan. Most of them belong to the 10th and 11th families of glycoside hydrolases. Families 5, 7, 8 and 43. Among them, most of the eighth family are cellulases, and also include some chitinases and xylanases. At present, there are only two xylanases in the eighth family that have undergone property research, namely pXyl and pXyl from Pseudoalteromonas.haloplanktis TAH3a Xyn8 from an environmental genome library. Based on the crystal structure analysis of pXyl, family 8 xylanases may fold into a twisted (a / a) 6 Barrel-like structure, surrounded by 6 a-helices in the outer and inner layers, this topology has also been used in family 9 endoglucanases, family 15 amylases...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10D21C9/10C12P19/14A23K1/00A21D8/04A23K20/189
Inventor 张玉忠郭兵陈秀兰解彬彬石梅张熙颖何海伦周百成
Owner SHANDONG UNIV
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