摘要
以超嗜热古菌Sulfolobus tokodaii strain 7基因组DNA为模板,通过PCR扩增高温酸性α-淀粉酶基因ST0817,将此基因克隆至表达载体pET15b,并转化大肠杆菌Escherichia coli BL21-CodenPlus(DE3)-RIL,获得重组大肠杆菌工程菌。通过热处理、镍柱亲和层析和分子筛层析,得到纯化重组酶,SDS-PAGE分析表明,该酶分子量为53.0 kDa。酶学性质研究表明,该酶最适温度和pH分别为75℃和5.5;具有较强的热稳定性和pH稳定性,在85℃处理8 h保持50%左右活力,在pH 5.2处理120 min仍保持50%活力。此酶对不同底物水解活性不同,直连淀粉>可溶性淀粉>支链淀粉>β-极限糊精>糖原>环糊精>普鲁兰糖;该酶对有机溶剂、变性剂和金属离子具有一定抗性。
A gene（ST0817） encoding a putative thermophilic and acidic α-amylase from the thermophilic crenarchaeota Sulfolobus tokodaii strain 7 was cloned and functionally overexpressed in Escherichia coli.The recombinant enzyme was purified to homogeneity after heat treatment,Ni-NTA affinity and superdex-200 gel filtration.The relative molecular mass of α-amylase was 53.0 kDa on SDS-PAGE.The purified enzyme displayed optimal activity at 70°C and pH 5.5.It had a half-life of 8 h at 85°C.The enzyme was found to have high pH stability and maintained about 50% of activity even after 120 min of treatment at pH 5.2.The enzymatic activities of the purified enzyme towards different substrates were listed from high to low as follows： amylose soluble starch amylopectin β-cyclodextrin glycogen cyclodextrin pullulan.The enzyme was found to have high tolerance to organic solvents,metal ions and detergents.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2013年第5期13-17,共5页
Food and Fermentation Industries
基金
国家自然科学基金项目(1176229)
河南省科技厅重点科技攻关项目(102102210063)
郑州市金水区科技攻关项目(20113142)
关键词
高温酸性α-淀粉酶
克隆
表达
酶学性质
thermophilic and acidic α-amylase
cloning
expression
enzymatic properties
