古细菌Pyrococcus furiosus高嗜热α-淀粉酶基因在大肠杆菌中的分泌表达

Secretory Expression of High Thermophilic α-amylase Gene of Pyrococcus furiosus, A Kind of Earchaic bacterium

该文将大肠杆菌表达载体pKK223-3中含tac启动子的片段以BamHI、EcoRI酶切后接入表达载体pET28a 中,构建成新的表达载体pEtac,通过PCR扩增获得P.furiosus 胞外α- 淀粉酶完整结构基因,接入pEtac 中,转化大肠杆菌JM109。在IPTG诱导下,转化子周质中能测出明显酶活,证明Pyrococcusfuriosus 的胞外α- 淀粉酶能在自身信号肽引导下分泌到大肠杆菌细胞周质中。重组酶最适pH为4.5,最适温度为95℃,重组酶经121℃保温1h,酶活仍能保持50%以上,性质与由P.furiosus自身分泌的胞外α- 淀粉酶相似。

Abstract This paper showed that the fragment containing the tac promoter in the expressive vector pKK223-3 of E. coli was inserted into the expressive vector Pet28a with the BamHⅠ、EcoRⅠ enzymatic cutting, which construct- ed the new expressive vetor pEtac. The gene with whole structure of P. furiosus extracellularα-amylase obtain- ed by the PCR expanding was transformed E. coli strain JM109 after being inoculated into pEatc. By induction of IPTG,The transformant could obviously check out the enzymatic activity, which proved that extracellular α-amylase of P. furiosus could secrete to cytoplasm of E.coli by signal peptide of itself. The optimum pH and temperature for recombinant enzyme were 4.5 and 95℃, respectively. Recombinant enzyme could maintain above 50% incubated for 1 h at 121℃. The character of recombinat enzyme was similar to extracellular α-amylase self-secreted from P. furiosus