This thesis investigates the effect of urea and guanidinium hydrochloride on the activity of recombinant pernisine and the relationship between changes in enzymatic activity and protein conformation. Pernisine is a thermostable extracellular serine protease from the hyperthermophilic archaeon Aeropyrum pernix and therefore represents an interesting model for studying enzyme function under challenging conditions. Protease activity was measured spectrophotometrically using azocasein at different temperatures and in the presence of various concentrations of the denaturants, whereas conformational changes were monitored by fluorescence spectroscopy and circular dichroism. The results showed that both denaturants increased pernisine activity, with the effect being more pronounced in the presence of urea. At the lower temperature, urea at selected concentrations led to activity comparable to that observed at the higher temperature without denaturant. Spectroscopic analyses indicated that the denaturants affected the local environment of tryptophan residues and caused partial changes in secondary structure, but did not induce extensive global denaturation. These findings suggest that the effect of denaturants on pernisine activity is more complex than structural changes alone and likely involves additional mechanisms.
|
