Proteolytic enzyme engineering : a tool for wool

One of the goals of protein engineering is to tailor the structure of enzymes to optimize industrial bioprocesses. In the present work, we present the construction of a novel high molecular weight subtilisin, based on the fusion of the DNA sequences coding for Bacillus subtilis prosubtilisin E and for an elastin-like polymer (ELP). The resulting fusion protein was biologically produced in Escherichia coli, purified and used for wool finishing assays. When compared to the commercial protease Esperase, the recombinant subtilisinE-VPAVG220 activity was restricted to the cuticle of wool, allowing a significant reduction of pilling, weight loss and tensile strength loss of wool fibers. Here we report, for the first time, the microbial production of a functionalized high molecular weight protease for controlled enzymatic hydrolysis of wool surface. This original process overcomes the unrestrained diffusion and extended fiber damage which are the major obstacles for the use of proteases for wool finishing applications

Secreted production of an elastin-like polypeptide by Pichia pastoris

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium