title: [Effect of N-terminal disulfide bridge on thermostability of family 11 xylanases].
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.
authors.affiliation: School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Gao
authors.forename: Junqing
authors.initials: J
authors.lastname: Wang
authors.forename: Minchen
authors.initials: M
authors.lastname: Wu
authors.forename: Cunduo
authors.initials: C
authors.lastname: Tang
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001236:Aspergillus oryzae / Q000201:enzymology; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases