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

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;Mn~(2+)、Co~(2+)、AL~(3+)、Fe~(3+)、SDS及EDTA对其酶活有不同程度的抑制作用;该酶的K_m、V_(max)、K_(cat)及K_(cat)/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).

Cloning,Expression and Enzymatic Characterization of the Pullulanase from an Extreme Thermophilic Bacteria Thermosipho melanesiensis

The pullulanase gene TM-pulA was choosed from the extreme thermophilic bacterium Thermosipho melanesiensis( DSM12029) and cloned by using the genome purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures as the template. The plasmid pET21 a containing TM-pulA was constructed by restriction enzyme digestion and linking method. The recombinant plasmid was transferred into strain Escherichia coli Rosetta( DE3). After purification,the hydrolysis products were analyzed and the enzymatic properties were determined. The results are as follows: TM-pulA is a type Ⅰ pullulanase; the optimum pH is 5.8; the optimum temperature is 80 ℃; The half-life is 4.75 h; Mn~(2+),Co~(2+),Al~(3+),Fe~(3+),SDS and EDTA inhibit the activity of enzymes in different degrees; the value of K_m,V_(max),K_(cat)and K_(cat)/Kmare 4.68 g·L~(-1),0.0085 mmol·L~(-1)·s~(-1),71.18 s~(-1) and 15.21 L·g~(-1)·s~(-1),respectively.