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极端嗜热菌Thermosipho melanesiensis普鲁兰酶基因的异源表达与酶学性质分析

Mingdao WANG,Yan XING,Shuang QIU,Hongyang WANG,Lipeng SUN,Liyou QIU

Journal of Xinyang Normal University(Natural Science Edition)（2018）

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Abstract
选择来源于极端嗜热菌Thermosipho melanesiensis (DSM12029) 的普鲁兰酶基因, 以购自德国菌种保藏中心的基因组为模板, 扩增出普鲁兰酶基因TM-pulA; 利用酶切酶连构建了重组质粒pET21a-TM-pulA; 转入大肠杆菌Rosetta (DE3) 菌株中诱导表达并经纯化后, 进行了水解产物分析和酶学性质测定. 结果显示: TM-pulA为Ⅰ型普鲁兰酶, 最适pH为5. 8; 最适温度是80 ℃; 70 ℃下半衰期为4. 75 h; Mn2+、Co2+、AL3+、Fe3+、SDS及EDTA对其酶活有不同程度的抑制作用; 该酶的Km、Vmax、Kcat及Kcat /Km值分别为4. 68 g·L-1、0. 0085 mmol·L-1·s-1、71. 18 s-1、15. 21 L·g-1·s-1.
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Key words
pullulanase,Thermosipho melanesiensis,heterologous expression,analysis of enzymatic properties
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