Strategies towards the functionalization of subtilisin E from Bacillus subtilis for wool finishing applications

Subtilisin E is an alkaline serine protease secreted by the Gram positive bacterium Bacillus subtilis and widely used in industry as a biocatalyst for various processes. The most common application of subtilisins is in laundry detergents. However, due to environmental concerns, the application of subtilisins to treat wool, is under study. There are some reports regarding the attempts to substitute the conventional chlorine treatment by an enzymatic process capable of providing the same characteristics to the fabric, like anti-shrinking and better uptake and fixation of the dyestuff. However, the degree of uncontrolled hydrolysis due to diffusion of the enzyme inside the wool fiber causes unacceptable losses of strength. To overcome this fact, and taking advantage of the x-ray crystallographic structure, the authors have modified subtilisin E genetically, increasing its molecular weight, to restrict the hydrolysis to the surface of the wool fibers. Therefore, three genetically modified enzymes with a molecular weight 2-fold to 4-fold higher than the native subtilisin E were produced and assessed for activity. The prokaryotic expression systems, pET25b(+), pET11b and pBADC, were explored for the production of recombinant enzymes. The results demonstrated that regardless the expression system or strain used, chimeric subtilisins were not expressed with the correct folding. No active and soluble recombinant protein was recovered under the testing conditions. Despite this drawback, a novel approach was described to increase the molecular weight of subtilisin. The reported results are noteworthy and can indicate good guidelines for future work aiming at the solubilization of recombinant chimeric subtilisins.