| Summary: | <p>Abstract</p> <p>Background</p> <p>Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, <it>Thermobifida fusca </it>cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in <it>Escherichia coli </it>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 <it>E. coli </it>to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.</p> <p>Results</p> <p><it>T. fusca </it>cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in <it>E. coli </it>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.</p> <p>Conclusions</p> <p>In the present study, <it>T. fusca </it>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 <it>E. coli </it>α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.</p>
|
|---|