| Sulfolobus tokodaii strain 7高温酸性α-淀粉酶基因在大肠杆菌中克隆表达及其酶学性质 |
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| 引用本文： | 魏涛,孙浩,申玉龙,毛多斌. Sulfolobus tokodaii strain 7高温酸性α-淀粉酶基因在大肠杆菌中克隆表达及其酶学性质[J]. 食品与发酵工业, 2013, 39(5): 13-17 |
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| 作者姓名： | 魏涛 孙浩 申玉龙 毛多斌 |
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| 作者单位： | 1. 郑州轻工业学院食品与生物工程学院,河南郑州,450002
2. 山东大学微生物技术国家重点实验室,山东济南,250100 |
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| 基金项目： | 国家自然科学基金项目，河南省科技厅重点科技攻关项目，郑州市金水区科技攻关项目 |
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| 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。
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| 关 键 词： | 高温酸性α-淀粉酶 克隆 表达 酶学性质 |
Cloning, over expression and characterization of a new thermophilic and acidic a-amylase from Sulfolobus tokodaii strain 7 in Escherichia coil |
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| WEI Tao,SUN Hao,SHEN Yulong,MAO Duobin. Cloning, over expression and characterization of a new thermophilic and acidic a-amylase from Sulfolobus tokodaii strain 7 in Escherichia coil[J]. Food and Fermentation Industries, 2013, 39(5): 13-17 |
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| Authors: | WEI Tao SUN Hao SHEN Yulong MAO Duobin |
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| Affiliation: | 1(School of Food and Biological Engineering,Zhengzhou University of Light Industry,Zhengzhou 450002,China) 2(State Key Laboratory of Microbial Technology,Shandong University,Jinan 250100,China) |
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| Abstract: | 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. |
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| Keywords: | thermophilic and acidic α-amylase cloning expression enzymatic properties |
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