Effect of N-terminal disulfide bridge on thermostability of family 11 xylanases.
- Author:
Shujuan GAO
1
;
Junqing WANG
;
Minchen WU
;
Cunduo TANG
;
Jing WU
Author Information
1. School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Amino Acid Substitution;
Aspergillus oryzae;
enzymology;
Base Sequence;
Disulfides;
chemistry;
metabolism;
Endo-1,4-beta Xylanases;
biosynthesis;
chemistry;
genetics;
Enzyme Stability;
genetics;
Molecular Sequence Data;
Mutagenesis, Site-Directed;
methods;
Pichia;
genetics;
metabolism;
Protein Engineering;
methods;
Recombinant Proteins;
biosynthesis;
chemistry;
genetics
- From:
Chinese Journal of Biotechnology
2012;28(12):1441-1449
- CountryChina
- Language:Chinese
-
Abstract:
A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
