Correlation between thermostability of the xylanase EvXyn11TS and its N-terminal disulfide bridge

Abstract:

Abstract:［Objective］ To reveal the correlation between thermostability of xylanase EvXyn11TS and its N-terminal disulfide bridge，an EvXyn11TS-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis．［Methods］Multiple homology alignment of protein primary structures between the EvXyn11TS and several GH family 11 xylanases displayed that，in their N-termini，only EvXyn11TS contained a disulfide bridge (Cys5 -Cys32)，whose effect on the xylanase thermostability was predicted by molecular dynamics simulation．We constructed a gene Syxyn11M，encoding the mutated xylanase (EvXyn11M ) without N-terminal disulfide bridge．Then，Syxyn11 and Syxyn11M 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 EvXyn11M was 70℃，which was 15℃ lower than that of EvXyn11TS．The half-life (t1/290) of EvXyn11TS at 90℃ was 32 min，while the t1/270 of EvXyn11M at 70℃ was only 8.0 min．［Conclusion］The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11TS 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.