title: [Enhancing stability of Trichoderma reesei xylanase (XYN II) by site-directed mutagenesis].
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.
authors.affiliation: Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
authors.forename: Chengye
authors.initials: C
authors.lastname: Han
authors.forename: Shiyuan
authors.initials: S
authors.lastname: Yu
authors.forename: Jia
authors.initials: J
authors.lastname: Ouyang
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Juan
authors.initials: J
authors.lastname: Zhou
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D019943:Amino Acid Substitution; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases