枯草芽胞杆菌fmbj SrfAC-A结构域的活性测定

Activity of the SrfAC-A domain from Bacillus subtilis fmbj

摘要:

摘要:【目的】在体外研究surfactin合成酶的A结构域，为获得新的surfactin类似物奠定基础。【方法】本文从枯草芽胞杆菌(Bacillus subtilis) fmbj中克隆出surfactin合成酶第7个模块的A 结构域基因(SrfAC-A)，与表达质粒pET-23a 相连后在大肠杆菌表达系统中表达，用Ni-NTA亲和柱对重组蛋白SrfAC-A进行分离纯化 后测定其活性。【结果】克隆所得的A结构域对Ile有选择活性，而对其他氨基酸基本无活性。【结论】Surfactin合成酶中的A结构域能在体外独立行使其选择底物氨基酸的功能。

关键词:关键词:Surfactin合成酶，A结构域，克隆表达，活性测定

Abstract:

Abstract:［Objective］ We studied the A domain of surfactin synthase in vitro to obtain new surfactin analogues．［Methods］We cloned the srfAC-A gene from Bacillus subtilis fmbj by PCR，and constructed a recombinant expression vector named pET-23a-srfAC-A． Furthermore，the SrfAC-A domain was expressed in E．coli BL21 (DE3) and purified by Ni-NTA agarose column． Then the activity of srfAC-A domain was detected． ［Results］The srfAC-A domain had specificity towards Ile，but almost no activity to other amino acids．［Conclusion］The independent A domain from surfactin synthase had selectivity to specific amino acids in vitro．

Key words:Keywords: Surfactin synthase，A domain，Cloning and expression，Activity analysis