Enhancing stability of Trichoderma reesei xylanase (XYN II) by site-directed mutagenesis.
- Author:
Chengye HAN
1
;
Shiyuan YU
;
Jia OUYANG
;
Xin LI
;
Juan ZHOU
;
Yan XU
Author Information
1. Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Substitution;
Disulfides;
chemistry;
metabolism;
Endo-1,4-beta Xylanases;
biosynthesis;
chemistry;
genetics;
Enzyme Stability;
genetics;
Mutagenesis, Site-Directed;
Pichia;
genetics;
metabolism;
Protein Engineering;
methods;
Recombinant Proteins;
biosynthesis;
chemistry;
Trichoderma;
enzymology;
genetics
- From:
Chinese Journal of Biotechnology
2010;26(5):623-629
- CountryChina
- Language:Chinese
-
Abstract:
We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50 degrees C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.
