Purification and Characterization of a Thermostable Protease from Pseudomonas aeruginosa NS-83

A bacterial strain NS-83 isolated from soil was able to produce an extracellular thermostable protease. The strain was identified as Pseudomonas aeruginosa based on its morphological and physiological characteristics. A thermostable protease from this strain has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification procedures included hydrophobic interaction, ion exchange, and gel filtration chromatography. The $M_r$ and the pl of the enzyme were 32,000 and 5.9, respectively. The optimal pH at 55$^{\circ}C$ and the optimal temperature at pH 7.0 were 8.0 and 60$^{\circ}C$, respectively. The D-values of the enzyme at 60, 65, and 70$^{\circ}C$ were 22, 2.1, and 0.75 hrs, respectively. The enzyme activity was significantly inhibited in the presence of 1 mM o-phenanthroline or EDTA, suggesting that the enzyme is metalloprotease. The $K_m$, and $V_{max}$ for Hammarsten casein were found to be 3.2 mg/ml and 0.918 unit/ml, respectively. These enzymatic properties were similar to those of elastase produced from P. aeruginosa IFO 3455, but the enzyme was clearly different from the reported elastase, in respect to $Ca^{++}$ effects on enzyme-thermostability. This property, together with amino acid composition analysis, confirmed that the enzyme differs from the known P. aeruginosa elastase.