近海温泉中嗜热菌Geobacillus sp. ZH1锰超氧化物歧化酶的克隆与表达  

Cloning and expression of manganese-containing superoxide dismutase from offshore hot spring Geobacillus sp. ZH1

作　　者：李鹤宾[1] 洪璇[1] 黄秀梅[1] 

机构地区：[1]厦门医学高等专科学校,福建厦门361008

出　　处：《台湾海峡》2012年第3期375-379,共5页Journal of Oceanography In Taiwan Strait

基　　金：福建省教育厅科技项目资助项目(JA11330);厦门医学高等专科学校校基金资助项目(K2011-3)

摘　　要：将嗜热菌Geobacillus sp. ZH1的超氧化物歧化酶(supseroxide dismutase,SOD)基因插入表达载体pET-32α(+),在Escherichia coli BL21(DE3)中进行表达,并利用亲和层析纯化重组超氧化物歧化酶.将制备的脱辅SOD进行Mn2+和Fe2+金属重构后,得到的Mn2+重构SOD的比活力达668U/mg,Fe2+重构Fe-SOD没有活性,说明ZH1菌株的超氧化物歧化酶为Mn-SOD.凝胶过滤及SDS-PAGE分析显示,Mn2+重构SOD为同聚二聚体,亚基分子量为71.7 kDa.这些研究结果为进一步研究该酶的生化特性及酶的定向进化研究奠定了良好的基础.The gene encoding a putative superoxide dismutase from thermophilic Geobacillus sp. ZH1 was cloned into the expression vector pET-32α（＋） and overexpressed in Escherichia coli BL21（DE3）.Recombinant superoxide dismutase was purified using affinity chromatography.The prepared apo-SOD was reconstituted with either Mn2＋ or Fe2＋ by means of incubation with appropriate metal salts.The molecular mass of Mn2＋-reconstituted SOD was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） analysis and size exclusion chromatography.Mn2＋-reconstituted SOD exhibited the specific activity of 668 U/mg and Fe2＋-reconstituted SOD had no specific activity which showed that SOD from strain ZH1 was Mn-SOD.The Mn2＋-reconstituted SOD was determined as homodimer with a monomeric molecular mass of 71.7 kDa.These results laid a foundation for further research of biochemical characteristics and directed evolution of this enzyme.

关 键 词：海洋生物学 超氧化物歧化酶 克隆 表达 重构 

分 类 号：P735[天文地球—海洋生物学]