| Title | Purification and Characterization of Two Extracellular Proteases from Oligotropha carboxydovorans DSM 1227 |
|---|---|
| Author | Kang, Beom Sik · Jeon, Sang Jun · Kim, Min Young * |
| Address | Molecular Microbiology Laboratory, Department of Biology, Yonsei University |
| Bibliography | Journal of Microbiology, 37(1),14-20, 1999, |
| DOI | |
| Key Words | Carboxydobacteria, extracellular protease, Oligotropha carboxydovorans |
| Abstract | Two extracellular proteases, EP I and EP II, from cells of Oligotropha carboxydovorans (formerly Pseudomonas carboxydovorans) DSM 1227 grown in nutrient broth were purified to greater than 95% homogeneity in five steps using azocasein as a substrate. The final specific activities of EPs I and II were 214.9 and 667.4 units per mg of protein. The molecular weights of native EPs I and II were determined to be 23,000. Sodium dodecyl sulfate-gel electrophoresis revealed the two enzymes to be monomers. The enzymes were found to be serine-type proteases. The activity of EP I was stimulated by Ca^2+, Mg^2+, and Ba^2+, but that of EP II was not. The enzymes were completely inhibited by Fe^2+, Hg^2+, Co^2+, Zn^2+, and Cd^2+. EDTA and EGTA exhibited a strong inhibitory effect on EP I. The optimal pH for the two enzymes was pH 9.0. The optimal temperatures for EP I and II were 60 and 50℃, respectively. The enzymes were stable under alkaline conditions. The thermal stability of EP I was higher than that of EP II. Cell-free extracts did not inhibit the purified enzymes. The enzymes were active on casein, azocasein, azocoll, and carbon monoxide dehydrogenase, but weakly active with bovine serum albumin. |
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