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Xylanase XynAS9-m mutants D185P/S186E with improved thermal stability as well as gene and application thereof

A technology of S186E and xylanase, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as poor thermal stability

Active Publication Date: 2015-04-22
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

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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  • Xylanase XynAS9-m mutants D185P/S186E with improved thermal stability as well as gene and application thereof
  • Xylanase XynAS9-m mutants D185P/S186E with improved thermal stability as well as gene and application thereof
  • Xylanase XynAS9-m mutants D185P/S186E with improved thermal stability 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 th...

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Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to xylanase XynAS9-m mutants D185P / S186E and V81P / G82E / D185P / S168E with improved thermal stability as well as genes and application thereof. Aspartic acid at the 185th site of xylanase which is shown by an amino acid sequence such as SEQ ID NO.1 is mutated into proline, and serine at the 186th site is mutated into glutamic acid to obtain a mutant; and further, valine at the 81st site is mutated into proline, glycine at the 82nd site is mutated into glutamic acid, the aspartic acid at the 185th site is mutated into proline, and the serine at the 186th site is mutated into glutamic acid to obtain mutants V81P / G82E / D185P / S186E. The thermal stability of two mutant enzymes is obviously improved, the optimal temperatures are respectively increased by 10 DEG C and 20 DEG C compared with 70 DEG C, the Tm values are respectively increased by 1.2 DEG C and 6.99 DEG C, and the potential application values of the xylanase XynAS9-m mutants in the industries of paper pulp brewing, biological energy sources and the like are revealed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a xylanase XynAS9-m mutant D185P / S186E 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, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12R1/84
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