HETEROLOGOUS EXPRESSION OF NOVEL PROTEASES

In view of the recent trend of developing environmentally friendly technologies the application of enzymes in until now environmentally harmful processes are coming more and more into focus. As biocatalysts, enzymes offer the large benefit that they work energy- and resource-efficient under comparatively mild conditions compared to the traditional chemical processes. Proteases belong to a large group of degradative enzymes that hydrolyze peptide bonds and account for about 60% of worldwide sales of industrial enzymes. They can be found in a variety of applications, e.g. as laundry detergent, as well as in leather, food and feed industry [1]. As constantly new application fields of these enzymes emerge, proteases with customized but robust characteristics are needed. In order to meet this demand, we underwent an approach to identify novel proteases by an in-silico search of genomes of putative protease-producing species, produce these candidate enzymes after optimization of the cultivation strategy in appropriate host organisms, E. coli and P. pastoris respectively, and determine their protein characteristics and enzymatic activities after purification. Some candidate proteases could not be produced in sufficient amount, most likely due to their intrinsic auto-catalytic activity resulting in fast degradation of the recombinant enzyme itself. Nevertheless, several proteases could finally be produced, which show high stability and activity over a broad pH and temperature range, making them promising enzymes for industrial applications.