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。
[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.
出处
《微生物学报》
CAS
CSCD
北大核心
2013年第4期346-353,共8页
Acta Microbiologica Sinica
基金
国家自然科学基金(31101229)~~
关键词
木聚糖酶
耐热性
N端二硫键
分子动力学模拟
定点突变
xylanase
thermostability
N-terminal disulfide bridge
molecular dynamics simulation
site-directed mutagenesis
