【摘要】 嗜热栖热菌(Thermus thermophilus)wl的超氧化物歧化酶基因在Escherichia coli BL21(DE3)中进行表达,采用Ni-NTA agarose亲和层析获得重组超氧化物歧化酶,比活力达565 U/mg.重组酶的相对分子质量为110.9 kDa,亚基相对分子质量为28.3 kDa,该酶为同聚四聚体蛋白.重组酶对氯仿-乙醇和SDS敏感,对H2O2不敏感,并且它的紫外最大吸收波长位于279 nm,判断其属于锰超氧化物歧化酶类型.1.0 mmol/dm3Mn2+对重组酶有激活作用,同浓度的其他金属离子Mg2+、Ca2+、Ba2+、Co2+、Cu2+、Zn2+和Fe2+对重组酶活力都有抑制作用,其中以Co2+的作用最强,抑制了约64%的酶活性.重组酶在70℃下处理1 h,保持原有活力的60%,即使在90℃下处理1 h,仍保持原有活力的35%.在pH值为4.0～11.0的缓冲液中25℃下处理1 h,保持65%以上的原始活力.由此可见,重组锰超氧化物歧化酶具有高的热稳定性和宽pH稳定性,在工业上具有巨大的应用潜力.更多还原

【Abstract】 The gene of the superoxide dismutase(SOD) from Thermus thermophilus wl was expressed in Escherichia coli BL21(DE3).The recombinant SOD was purified by Ni-NTA resins affinity chromatography and then characterized.Results: The recombinant enzyme had the specific activity of 565 U/mg.It was a homotetramer with a monomeric molecular weight of 28.3 kDa and total molecular weight of 110.9 kDa.The recombinant SOD was sensitive to Chloroform-ethanol and SDS,while insensitive to H2O2.The ultraviolet absorption peak of the enzyme was found at 279 nm.The results indicated that the recombinant SOD was of the Mn-SOD type.The enzymatic activity was enhanced by 1.0 mmol/dm3 Mn2+,while it was inhibited by Mg2+,Ca2+,Ba2+,Co2+,Cu2+,Zn2+ and Fe2+ at the same concentration.The effect of Co2+ was the most negative with an inhabitation ratio of about 64%.The recombinant enzyme retained 60% of its initial activity after incubation at 70℃ for 1 h.Even after heating the enzyme at 90℃ for 1 h,35% of the initial activity was maintained.This enzyme retained over 65% of the initial activity,after incubation at 25℃ for 1 h across a pH span from 4.0 to 11.0.Conclusion: Thermus thermophilus wl recombinant Mn-SOD had good stability at high temperatures and wide pH range,and thus it had considerable potential applications for various industrial purposes.更多还原