Xylanase Scxyn5 as well as coding gene and application thereof

A xylanase and gene technology, applied in the field of genetic engineering, can solve problems such as different physical and chemical properties and biological functions, and achieve the effects of reducing costs, reducing use, and preventing damage

Pending Publication Date: 2022-04-12
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are no reports on the application of GH10 family and GH11 family extreme halophilic/halat-tolerant xylanase to hydrolyze corn fiber glue in high-salt system to prepare

Method used

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  • Xylanase Scxyn5 as well as coding gene and application thereof
  • Xylanase Scxyn5 as well as coding gene and application thereof
  • Xylanase Scxyn5 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning of the gene encoding xylanase Scxyn5 of embodiment 1

[0036] Schizophyllum commune was fermented and cultured with corn bran fiber as a carbon source, and the mRNA was isolated from the bacteria for transcriptome sequencing analysis; the Scxyn5 coding gene (Xyn5-F: 5′-CCG GAATTC CTCCCCAAGCGTCAGACCACT-3′; Xyn5-R: 5′-CGCTA GCGGCCGC TGCACCCAAG CCGCTCAC-3'), and constructed the recombinant vector Scxyn5-pET28a.

Embodiment 2

[0037] The construction of embodiment 2 xylanase recombinant vectors

[0038]Using the xylanase Scxyn5-pET28a derived from S.commune sp.DB1 as a template, the PCR reaction was used to clone the Scxyn5 fragment of the xylanase gene. The primers and reaction conditions were as follows:

[0039] Scxyn5-F: 5′-CCG GAATTC CTCCCC AAGCGTCAGCCACT-3′;

[0040] Scxyn5-R: 5′-CGCTA GCGGCCGC TGCACCCAAG CCGCTCAC-3';

[0041] The amplified products were digested by EcoRI and NotI, respectively, and directly ligated with the cut pPIC9k plasmid, transformed into TransI-T1 competent cells, and the transformants were picked and sequenced for verification. The correct transformant verified by sequencing was the recombinant yeast expression plasmid pPIC9K-Scxyn5.

Embodiment 3

[0042] Example 3 Construction of the Pichia pastoris engineering bacteria recombinantly expressing the xylanase gene

[0043] The recombinant expression vector pPIC9K-Scxyn5 was linearized with endonuclease SacI and transformed into Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / pPIC9K-Scxyn5.

[0044] Pick the positive transformant and transfer it to a 50mL Erlenmeyer flask containing 5mL of BMGY medium, place it at 30°C, and culture it on a shaker at 230rpm for 36h; centrifuge the fermentation broth at 3000g for 5min, discard the supernatant and use 5mL of BMMY medium to precipitate the bacteria Resuspended, then placed at 30°C, 230rpm shaker induction culture for 72h. Such as figure 1 As shown, the supernatant of the fermentation broth was used for enzyme activity detection and SDS-PAGE electrophoresis detection.

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Abstract

The invention relates to the field of gene engineering, in particular to xylanase Scxyn5 and a coding gene and application thereof. The amino acid sequence of the gene is as shown in SEQ ID NO: 1. The invention provides xylanase Scxyn5 of Schizophyllum commune, the xylanase Scxyn5 is an extremely salt-tolerant/halophilic xylanase, and a high-salt-concentration catalytic system can improve the hydrolytic activity of the xylanase to corn viscose, so that the corn viscose can be degraded by the xylanase Scxyn5, and the corn viscose can be degraded by the xylanase Scxyn5, so that the corn viscose can be degraded by the xylanase Scxyn5. Arabinoxylan with different component force distribution characteristics can be prepared by applying xylanase single enzyme to hydrolyze corn viscose, and a basis is provided for industrially preparing functional arabinoxylan with specific molecular weight by taking corn bran as a raw material.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to xylanase Scxyn5 and its coding gene and application. Background technique [0002] Endo-β-1,4-xylanase (β-1,4-xylanase, EC 3.2.1.8) is a glycoside hydrolase that catalyzes the hydrolysis of xylan backbone β-1,4-D- Xylosidic bonds generate xylooligosaccharides, which are the most important class of glycoside hydrolases in the xylan degrading enzyme system. Based on the sequence conservation of the catalytic domain, xylanases are classified as glycoside hydrolases (GH) No. 5, 7, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, 62 family. At present, most of the reported xylanases are distributed in the GH10 family and the GH11 family. Halophilicity / salt tolerance is an important property of enzymes. Halophilic / halt-tolerant xylanases generally refer to xylanases that maintain catalytic activity at high salt concentrations (0.5-4.5M). Halophilic / halal-tolerant xylanase has great app...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12P19/14C12R1/84
Inventor 刘玉春王超任菲张维清
Owner ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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