title: [Correlation between thermostability of the xylanase EvXyn11(TS) and its N-terminal disulfide bridge].
abstract: OBJECTIVE
  To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis.
  
  
  METHODS
  Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed.
  
  
  RESULTS
  The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min.
  
  
  CONCLUSIONS
  The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.
authors.affiliation: School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China. minrou.jiangnan@163.com
authors.forename: Rou
authors.initials: R
authors.lastname: Min
authors.forename: Jianfang
authors.initials: J
authors.lastname: Li
authors.forename: Shujuan
authors.initials: S
authors.lastname: Gao
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Minchen
authors.initials: M
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D004220:Disulfides; D043364:Endo-1,4-beta Xylanases