超嗜热细菌Thermotoga maritima酯酶Tm1350克隆、表达及其酶学性质研究

Cloning,expression and characterization of the esterase Tm1350 from hyperthermophilic bacteria Thermotoga maritima

摘要: 以超嗜热细菌Thermotoga maritima基因组DNA为模板,通过PCR法扩增酯酶Tm1350基因,构建重组质粒p ET15b-Tm1350,在大肠杆菌Escherichia coli BL21（DE3）中实现表达,并采用p-NPA方法测定其酶活性。研究结果表明,酯酶Tm1350可以水解不同碳链长度（10C）的对硝基苯酚酯,其中对硝基苯酚戊酸酯（p NP-C5）是该酶最适合底物;该酶最适反应温度和p H分别为70℃和6.5,90℃处理5h,仍有50%以上的酶活力,具有较强的热稳定性。酯酶Tm1350对金属离子、抑制剂、变性剂和有机溶剂具有较强的抗性。

Abstract: The esterase gene Tm1350 from Thermotoga maritima was amplified by PCR method and recombinant plasmid p ET15b- Tm1350 was constructed to express the enzyme in Escherichia coli BL21( DE3).Esterase activity was determined spectrophotometrically by the p- NPA method using p- nitrophenyl esters of various chain lengths as the substrates.The enzyme could hydrolyze the substrates of p NP- esters with acyl chains of different lengths and exhibited the highest activity for p NP- C5 among the substrates tested. Tm1350 displayed optimal activity at70℃ and 6.5.It maintained above 50% activity after 5h incubation at 90℃,which indicated that the enzyme showed strong thermal stability. The recombinant esterase Tm1350 was found to have strong resistances to metal ions,inhibitor,denaturant and organic solvents.