Isolation and Biochemical Characterization of Thermophilic Alkaline Proteases Resistant to Surfactant and Chelator from Bacillus sp. GUS1

Bacillus sp. GUS1, isolated from soil of the citrous garden in Iran was screened on gelatin agar medium for their ability to produce alkaline protease. The zymogram analysis of partial purification of the enzymes showed the presence of at least three proteases. The enzymes were active in a wide pH range (6.0-12.0) and stable in the alkaline range (8.0-12.0). The optimum temperature for the proteases was 70 °C. The irreversible thermoinactivation of the proteases showed that the enzymes retained 100% of its original activity after 60 min of incubation at 70 °C and, therefore, the proteases are highly stable at 70 ºC. Enzymes were mostly inhibited by PMSF, while the enzyme activity was retained to about 90% and 80% in the presence of 2-mercaptoethanol and iodoacetate, respectively. These data show that most of these proteases seem to be serine alkaline protease. Addition of SDS also marginally influenced the protease activity. Enzyme activity was remained more than 90% and 80% in the presence of 1 and 10 mM EDTA respectively. On the other hand, adding Ca2+ did not influence the enzyme activity. Our results suggested that the enzymes are not metalloproteases and are Ca2+-independent.