木聚糖酶EvXyn11TS耐热性与其N端二硫键的相关性分析

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

摘要:

摘要:【目的】以糖苷水解酶11 家族耐热木聚糖酶EvXyn11TS为研究对象，定点突变其编码基因Syxyn11，揭示EvXyn11TS耐热性与其N端二硫键的相关性。【方法】对不同来源的、与EvXyn11TS一级结构相似度较高的若干11家族木聚糖酶进行多序列同源比对，发现只有耐热的EvXyn11TS在其N端存在一个二硫键(Cys5－Cys32);运用分子动力学模拟预测该N端二硫键存在与否对木聚糖酶热稳定性的影响。以人工合成的Syxyn11为母本，采用PCR技术将其编码Cys5的密码子TGT突变为编码Thr5的ACT，构建去除了N端二硫键的突变酶(EvXyn11M)的编码基因Syxyn11M;分别将Syxyn11和Syxyn11M在毕赤酵母GS115中进行表达，并分析表达产物EvXyn11TS和EvXyn11M的温度和pH特性。【结果】酶学性质研究结果表明:EvXyn11M的最适温度Topt由突变前的85℃降至70℃;EvXyn11TS在90℃的半衰期t1/290为32 min，而EvXyn11M在70℃的半衰期t1/270仅为8.0 min。【结论】运用分子动力学模拟预测了N端二硫键对EvXyn11TS耐热性的重要作用，并通过定点突变验证之，为其它与耐热EvXyn11TS一级结构相似的、11家族常温高比活性木聚糖酶的耐热性改造提供了新的技术策略。

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.