BackgroundExtracellular 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.ResultsT. 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.ConclusionsIn 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.

Near-field electrospinning has been demonstrated to be able to achieve direct-write and highly aligned chitosan nanofibers (CNF) with prescribed positioning density. Cell spreading in preferential direction could be observed on parallel-aligned nanofibers, and the CNF patterns were capable of guiding cell extension when the distances between them are 20 and 100 μm, respectively. Alignment of the cells was characterized according to their elongation and orientation using the fast Fourier transform data and binary image analysis. Parallel CNF indicates that the alignment values sequentially increased as a function of positioning density such that incrementally more aligned cells were closely related to the increasing CNF positioning density. These maskless, low-cost, and direct-write patterns can be facily fabricated and will be a promising tool to study cell-based research such as cell adhesion, spreading, and tissue architecture.

No abstract