嗜热革节孢第十二家族内切葡聚糖酶的定点突变.docx

嗜热革节孢第十二家族内切葡聚糖酶的定点突变 摘要：纤维素酶有广泛的应用前景，探究纤维素酶的性质及其催化作用机制有重要意义。本研究对嗜热革节孢第十二家族内切葡聚糖酶steg1基因进行定点突变，研究突变对酶活性的影响。选择位于酶催化活性通道上的6个氨基酸位点（N35、W37、Y77、W128、Y145、N168）进行突变，得到8个突变酶（N35Q、W37H、Y77F、W128S、Y145F、Y145A、Y145W、N168Q）。结果显示，与野生酶WT相比，所有突变酶的活性降低，其中突变酶Y77F、W128S、Y145A的Km值和kcat值都降低；N35Q的Km和kcat值都升高；W37H、Y145F、Y145W、N168Q的Km值升高，kcat值降低。WT与突变酶的最适pH值为5.0，均保持不变。突变酶Y77F、N35Q的最适温度降低到50℃，WT和其余突变酶的最适温度为55℃。突变酶Y77F和N35Q在60℃处理10 min后分别保持49.34%和22.90%的活性，WT和其余突变酶在60℃处理10 min后都失活。本研究为探究内切葡聚糖酶的催化作用机理以及分子改造奠定基础。 关键词：内切葡聚糖酶；定点突变；酶活性；酶学性质 中图分类号：S188+.3文献标识号：A文章编号：1001-4942（2018）07-0055-07 Abstract Cellulase has a wide application prospect， so it is of great significance to explore the properties of cellulase and its catalytic mechanism. Site-directed mutagenesis of the steg1 gene of endoglucanase in the 12th glycoside hydrolase family of Scytalidium thermoohilium was carried out to study the effect of mutation on the enzyme activity. Six amino acid sites （N35，W37，Y77，W128，Y145，N168） located on the catalytically active channel were selected and mutated to obtain eight mutant enzymes （N35Q， W37H， Y77F， W128S， Y145F， Y145A， Y145W， N168Q）. The results showed that the activities of all mutants decreased compared with WT； the Km and kcat value of mutant Y77F， W128S and Y145A all decreased； the Km and kcat value of N35Q both increased； the Km value of W37H， Y145F， Y145W and N168Q increased and the kcat value decreased. The optimum pH of WT and mutant enzyme was 5.0， both of which remained unchanged. The optimum temperature of mutant Y77F and N35Q decreased to 50℃， and the optimum temperature of WT and other mutants was 55℃. Mutant enzyme Y77F and N35Q remained 49.34% and 22.9% activity after being treated at 60℃ for 10 minutes respectively， while WT and the rest mutants were all inactive. The research laid foundations for exploring catalytic mechanism of endoglucanase and the molecular modification. Keywords Endoglucanase； Site-directed mutagenesis； Enzymatic activity； Enzymatic property 纤维素酶是指能水解