嗜热栖热菌重组锰超氧化物歧化酶的性质  

Characterization of a recombinant manganese-superoxide dismutase from Thermus thermophilus

作　　者：李鹤宾[1,2] 席守民[2] 杨五彪[2] 叶子坚[1] 

机构地区：[1]厦门医学高等专科学校,福建厦门361008 [2]河南科技大学医学院,河南洛阳471003

出　　处：《台湾海峡》2012年第1期53-57,共5页Journal of Oceanography In Taiwan Strait

基　　金：厦门市科技计划资助项目(3502Z20093041);河南科技大学青年科学基金资助项目(2010QN0023)

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.The gene of the superoxide dismutase （SOD） from Thermus thermophilus wl was expressed in Escherichiacoli BL21 （ DE3 ）. The recombinant SOD was purified by NiNTA resins affinity chromatography and then characterized. Results ： The recombinant enzyme had the specific activity of 565 U/mg. It was a homotetramer with a monomerit 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 H202. The ultraviolet absorption peak of the enzyme was found at 279 nm. The results indicated that the recombinant SOD was of the MnSOD type. The enzymatic activity was enhanced by 1.0 mmol/dm3 Mn^2 ＋ , while it was inhibited by ng^2 ＋ , Ca^2 ＋ , Ba^2 ＋ , Co^2 ＋ , Cu^2 ＋ , Zn^2 ＋ and Fe^2 ＋ at the same concentration. The effect of Co^2＋ 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 MnSOD had good stability at high temperatures and wide pH range, and thus it had considerable potential applications for various industrial purposes.

关 键 词：海洋生物学 锰超氧化物歧化酶 热稳定 嗜热栖热菌wl 

分 类 号：P735[天文地球—海洋生物学]