Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3)
1 State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave, Wuxi, Jiangsu 214122, China
2 School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Ave, Wuxi, Jiangsu 214122, China
3 Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
Microbial Cell Factories 2012, 11:8 doi:10.1186/1475-2859-11-8Published: 12 January 2012
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.
T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase.
In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.