Xylanase mutant with improved specific enzymatic activity and coding gene and application thereof

A xylanase mutation and coding gene technology, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of low specific enzyme activity, instability, high cost, etc., and achieve high enzyme activity, action temperature and pH range wide effect

Active Publication Date: 2016-12-07
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the currently used xylanase has problems such as low specific enzyme activity, instability, high cost, and cannot meet the production needs, so it is necessary to optimize the properties through molecular modification

Method used

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  • Xylanase mutant with improved specific enzymatic activity and coding gene and application thereof
  • Xylanase mutant with improved specific enzymatic activity and coding gene and application thereof
  • Xylanase mutant with improved specific enzymatic activity and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Error-prone PCR amplification gene xynHBN3

[0045] The information of xynHBN3 can refer to the patent literature with the patent publication number CN1924002A; the plasmid pHBM130 is used as a template (preserved by the Key Laboratory of Industrial Biotechnology in Hubei Province, which can be obtained by the public, and there is a xylanase gene on the plasmid. For details, see Zhang Guimin's doctoral thesis "Cloning, Expression and Enzymatic Properties of Xylanase Gene"), using primer 18N3F (sequence: 5'CGC GGATCC GCGGAAACG3') and 18N3R (sequence: 5'ACGC GTC GAC CTTTTATC3') for PCR amplification, the primers added a BamH I restriction site upstream of the gene, and a Sal I restriction site downstream, the PCR amplification conditions were 94°C, 5min; 94°C, 30s, 58°C, 30s, 72°C, 45s, 30 cycles; 72°C extension for 10min. After recovering the PCR product by 1% agarose gel electrophoresis, BamHI and SalⅠ double digestion. At the same time, the pET28a plasmi...

Embodiment 2

[0055] Embodiment 2 Construction of mutant xylanase recombinant expression vector

[0056] 1. Double digestion plasmid vector and gene fragment

[0057] Take 5 μg of the pET28a vector and 5 μg of the error-prone PCR product and digest them with Sal Ⅰ and BamH Ⅰ, respectively, and divide them into 5 tubes for enzyme digestion. The system for each tube is as follows:

[0058] Sal I

3μL

BamH Ⅰ

3μL

10×digestion buffer

10 μL

Error prone PCR product or pET28a vector

1μg

wxya 2 o

Up to 100μL

[0059] The reaction system was placed in a 1.5mL centrifuge tube and placed in a 37°C water bath for 3 hours. After detection by agarose electrophoresis, the carrier and error-prone PCR products were gel-recovered separately. The recovered products required A260 / A230>2.0 and A260 / A280≈1.8.

[0060] 2. Recombinant plasmid connection

[0061] Add the error-prone PCR-amplified target fragment (abbreviated as ep-PCR product) recovere...

Embodiment 3

[0070] The screening of embodiment 3 xylanase mutant libraries

[0071] A total of about 20,000 transformants were obtained after the electric transformation of the recombinant plasmid in Example 2. Spot all the transformants onto a xylanase activity screening plate (0.5% cross-linked xylan, 1% peptone, 1 % sodium chloride, 0.5% yeast powder, 1.5% agar), and the culture temperature was 37°C, from which 46 mutants whose transparent circle was significantly larger than the original strain transparent circle were selected. First screened by the hydrolysis circle method, and then re-screened by expanding the culture of E. coli shake flasks to induce expression. figure 1 Finally, a strain with the highest enzyme activity was screened and its gene was named xynHBN3217.

[0072] For the preparation method of cross-linked xylan, please refer to the literature: Expression of xylanase XYNZG in Kluyveromyces lactis and its application in bread baking, Zhan Feixiang, Hubei University, 2...

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Abstract

The invention relates to a xylanase mutant with improved specific enzymatic activity and a coding gene and application thereof. The xylanase mutant a protein shown in (a), (b) or (c): (a), a protein composed of amino acid sequences shown in SEQ ID NO. 1; a protein derived from (a) by subjecting the amino acid sequences shown in SEQ ID NO. 1 to substitution, deletion and / or addition of one or more amino acid residues and which has xylanase activity; (c), a protein coded by subjecting the amino acid sequences of code (a) and the amino acid sequences of code (b) to molecular hybridization and which has xylanase active amino acid sequences. The xylanase mutant according to the invention has specific enzymatic activity increased by 2.8 times, is applicable to degrading xylan substrates, has a wide range of acting temperature and pH, and has the advantages such as good resistance to acids and bases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a xylanase mutant with improved specific enzyme activity, its coding gene and its application. Background technique [0002] Xylanase can be widely used in brewing, feed, paper pulp, food, textile, energy and other industries. Xylanase refers to the general term for a group of enzymes that can degrade xylan into oligosaccharides and xylose, mainly including exo-β-1,4-xylanase, endo-β-1, 4-Xylanase and β-Xylosidase. Xylanase can decompose feed non-starch polysaccharides (NSPS) into xylo-oligosaccharides with a smaller degree of polymerization, thereby improving feed performance, eliminating or reducing the anti-nutrition caused by high viscosity of non-starch polysaccharides in the animal's stomach effect. At the same time, it can destroy the structure of plant cell walls, increase the activity of endogenous digestive enzymes, and improve the utilization of feed nutrients. In the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12P19/14C12P19/04
CPCC12N9/248C12N9/2482C12P19/04C12P19/14C12Y302/01008C12Y302/01037
Inventor 张桂敏卢毅宏王钦宏周玉玲马延和
Owner HUBEI UNIV
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