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对来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB进行同源建模,并结合嗜热木聚糖酶氮末端芳香族氨基酸疏水作用的结构分析,设计了XYNB的T11Y定点突变,观察XYNB分子中折叠股B1和B2的疏水作用对酶的热稳定性的影响。将突变酶XYNB′在毕赤酵母中表达,表达的XYNB′经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,XYNB′的耐热性比XYNB有明显的提高,但最适温度与原酶一样为6 0℃。另外,XYNB′的最适pH、Km值及比活性均有一定的改变。实验证实了木聚糖酶XYNB的氮端芳香族氨基酸之间的疏水相互作用与其热稳定性相关,为进一步的结构与功能研究提供了优良的基因材料。
Homologous modeling of high specific activity xylanase XYNB from Streptomyces olivaceoviridis and the structural analysis of the hydrophobic interaction of the aromatic amino acid nitrogen end of thermophilic xylanase were used to design the T11Y site-directed mutagenesis of XYNB. Effect of Hydrophobic Interaction of Folding Strands B1 and B2 on the Thermostability of Enzymes. XYNB ’was expressed in Pichia pastoris. The expressed XYNB’ was purified and compared with the original enzyme XYNB (purified by Pichia pastoris) for enzymatic properties. The results showed that the heat resistance of XYNB ’ There is obvious improvement, but the optimum temperature is the same as the original enzyme 60 ℃. In addition, the optimum pH, Km value and specific activity of XYNB ’are all changed to some extent. The experiment confirmed that the hydrophobic interaction between the nitrogen-terminal aromatic amino acids of xylanase XYNB was related to its thermal stability, which provided an excellent genetic material for further structural and functional studies.