Enzymes from thermophilic organisms are desired in industrial applications, due to their exceptional stability. One of the industrially relevant enzymes is pernisin from the hyperthermophilic archaeon Aeropyrum pernix. Pernisin is a subtilisin-like proteinase that stands out among other proteinases due to its prion protein degrading activity. Therefore, pernisin might be used for cleaning different surfaces contaminated with prions, as well as in food and leather industry.
As for other subtilisins, pernisin is synthesized in its inactive proform, which is then autocatalitically converted into the active proteinase. In this thesis we investigated the activation process of propernisin. We prepared different propernisin mutants and analyzed their folding and activation capability. We found out that propernisin activation proceeds in several steps. First, the peptide bond between the propeptide and the catalytic domain is autocatalitically cleaved, resulting in proteolytically inactive non covalent complex of the propeptide and the catalytic domain. The activation is completed upon dissociation of the autoprocessed complex and degradation of the released propeptide with the catalytic domain. For this final activation step, calcium ions are needed since they enable pernisin to fold into ordered conformation, which is protected against autodegradation. Importantly, the unique insertion that forms an additional calcium ion binding site within the catalytic domain is essential for pernisin stability during the activation. Besides calcium ions, also temperatures above 80 °C are required for the complete activation, as the released propeptide is destabilized at high temperatures only, which eventually leads to propeptide degradation. The major role of the pernisin propeptide is inhibition of the catalytic domain during the activation. Interestingly, the propeptide did not show any chaperoning activity, which is commonly observed in bacterial subtilisins.
We also optimized preparation of recombinant pernisin in Escherichia coli. Aggregation of propernisin in cytoplasm was prevented by translocation of produced propernisin in periplasmic space. Consequently, this strategy simplified isolation procedure of this proteinase. Pernisin that was isolated from the periplasm was highly thermostable and active. Additionally, different N-terminally and C-terminally truncated variants of pernisin were prepared and analyzed for their proteolytic activities. Based on our results, the outer regions of the catalytic domain with calcium binding sites are important for pernisin thermostability.