Abstract:
The thesis describes the isolation, purification and characterization of two extracellular thermophilic proteases from the strains S. thermovulgaris and Paenibacillus sp.
Both strains were obtained from Nigerian soil and showed good growing features. The exoenzymes were effectively purified by using a fractionated ammonium sulfate precipitation followed by a ion-exchange and size exclusion chromatography, respectively.
The molecular mass was examined by SDS-PAGE. For the proteases isolated from Paenibacillus sp. a molecular mass of 18 kDa , and for the proteases from S. thermovulgaris, a value of 24 kDa was obtained.
The specificity of both proteases were characterized by testing several synthetic substrates and specific inhibitors. As a result both proteases showed chymotrypsin-like specifity.
In addition the inhibition with phenylmethanesulfonylfluoride and 4-(2-aminoethyl)-benzene-sulfonylfluoride proved the existence of serine proteases with chymotrypsin-like specifity. Other inhibitors for other types of proteases did not affect their activity.
The temperature- and pH-stability of both proteases were investigated. The optimum pH and temperature values for enzyme activity of the protease from S. thermovulgaris and Paenibacillus sp. were examined at pH 9-10, Tmax=65°C, and pH 8-9, Tmax =45°C, respectively.
Finally, the possibility for technical use were investigated by immobilization of the enzymes.