Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof

A kind of xylanase mutation, xylanase technology, applied in the field of genetic engineering and enzyme engineering, can solve problems such as poor thermal stability

Active Publication Date: 2013-10-09
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, the optimum temperature of most xylanases is 45-55°C, and their thermal stability is poor, which cannot meet the requirements in pulp brewing, and high-temperature xylanases or heat-resistant enzymes can reduce the cost of enzyme preparations and improve reaction catalysis. High efficiency, reduced reaction energy consumption, reduced contamination of bacteria and high tolerance to chemical denaturants and related metal interest

Method used

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  • Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof
  • Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof
  • Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof

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Experimental program
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Embodiment 1

[0139] 1. Acquisition of mutant genes:

[0140] The gene sequence (SEQ ID NO.2) of xylanase XynAS9-m derived from Streptomyces sp.S9 was transformed, the mutation was introduced by means of Overlap PCR, and it was sequenced to obtain the mutant gene.

[0141] The mutation includes six PCR primers: S9BF, S9BR, V81P / G82E-F, V81P / G82E-R,

[0142] D185P / S186E-F, D185P / S186E-R.

[0143] The primer sequences are as follows:

[0144] S9BF:5'-TA GAATTC GACACCGCCACCCTGGGCGAACT-3'

[0145] S9BR: 5'-TAT GCGGCCGC CTACGCCGAAGTCCCGGACGGC-3'

[0146] V81P / G82E-F:5'-GCCAGATCACCcccgaaAACACCATGAAGT-3'

[0147] V81P / G82E-R:5-ACTTCATGGTGTTttcgggGGTGATCTGGC-3'

[0148] D185P / S186E-F:5'-AGAAGATCGGCcccgagTACATCG-3';

[0149] D185P / S186E-R:5'-CGATGTActcgggGCCGATCTTCT-3'

[0150] The underline represents the restriction enzyme cutting sites EcoRI and NotI, and the lowercase letters represent the mutant bases. The overlapping extension PCR method is completed through 3 PCR reactions. Take the...

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Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering and particularly relates to thermal stability improved xylanase XynAS9-m mutants V81P/G82E and V81P/G82E/D185P/S186E as well as a gene and application thereof. The xylanase XynAS9-m mutant V81P/G82E is a xylanase with an amino acid sequence shown as SEQ ID No.1, wherein the valine on the 81st position of the xylanase is mutated into proline, and the glycine on the 82nd position of the xylanase is mutated into glutamic acid. Furthermore, the valine on the 81st position of the xylanase is mutated into the proline, and the glycine on the 82nd position of the xylanase is mutated into the glutamic acid; and the aspartic acid on the 185th position of the xylanase is mutated into the proline, and the serine on the 186th position of the xylanase is mutated into the glutamic acid, so that the xylanase XynAS9-m mutant V81P/G82E/D185P/S186E is obtained. The thermal stability of the mutated enzyme obtained by the invention is remarkably improved, so that the mutant has a potential application value in industries such as paper pulp making, biological energy source and the like.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and specifically relates to a xylanase mutant XynAS9-mV81P / G82E with improved thermostability and its gene and application. Background technique [0002] Xylanase (endo-1,4-β-xylanases, EC3.2.1.8) is an important class of industrial enzymes, a general term for a class of enzymes that degrade xylan into oligosaccharides and xylose. It mainly hydrolyzes the β-1,4-glucosidic bonds in xylan molecules in an endo-cutting manner to generate xylooligosaccharides and xylose. It is one of the most critical hydrolytic enzymes in the hemicellulolytic enzyme system. Most of the glycanases belong to the F / 10 and G / 11 families, and the xylanases of the tenth family have lower substrate specificity, faster hydrolysis and lower polymerization degree of hydrolyzate than those of the eleventh family. Therefore, it has important application value in industry. In recent years, xy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2482
Inventor 姚斌罗会颖王坤王亚茹孟昆石鹏君黄火清柏映国杨培龙赵珩马锐
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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