Thermotoga maritima普鲁兰酶的基因克隆与酶学性质研究

以海栖热袍菌(Thermotoga mariti ma)MSB8基因组DNA为模板,PCR扩增出普鲁兰酶基因pulA,克隆入表达载体pET28a,转化EscherichiacoliBL21-CodonPlus(DE3)-RIL,经IPTG诱导,测定普鲁兰酶酶活性。结果表明,重组转化子的细胞破碎液有普鲁兰酶活性,SDS-PAGE电泳结果显示出分子量约为96ku特异性蛋白质条带。酶学性质分析表明,其最适反应温度达到95℃,在30~80℃均保持最大酶活力的80%以上,最适反应pH值为6.0,且在碱性条件下稳定。

Thermotoga maritima MSB8 Pullulanase: Gene Cloning, Heterologous Over-expression and Biochemical Properties

The gene pulA was amplified by the method of PCR with the template of the genomic DNA of Thermotoga maritima MSB8,and cloned into the expression vector,pET28a,yielding hybrid plasmid pET28a-pulA.Subsequently,pET28a-pulA was introduced into Escherichia coli BL21-CodonPlus(DE3)-RIL and was successfully expressed through the activity of pullulanase.The recombinant protein expressed in E.coli showed extreme thermostability and pH stability with the most activity at 95℃ and pH 6.0.It was shown to be stable over the temperature range of 30～80 ℃ and the pH range of pH 6.0～8.0.