Nutrient broth에서 성장한 Acinetobacter sp. strain JC1으로부터 azocoll을 기질로 사용하여 95%이상의 순도를 지닌 두 가지 단백질 가수분해효소(protease I과 II)를 분리, 정제하였다. 두 효소의 최종 specific activity는 protease II가 protease I보다 약 27배 높았으며, 분자량은 protease I이 55,000이고 protease II가 44,000인 것으로 밝혀졌고, 두 효소 모두 한 개의 소단위로 구성되어 있음이 확인되었다. 정제된 두 효소는 serine-type의 단백질 가수분해효소로 minimal medium에서 성장한 세균에서는 형성되지 않았다. Protease I의 활성은 $Ca^{2+}$에 의해 증가되었으나 protease II는 영향을 받지 않았다. 두 효소는 모두 $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Fe^{2+}$, EDTA에 의해 활성이 완전히 억제되었으나 EGTA에 의해서는 영향을 받지 않았고, EDTA에 의해 억제된 protease II는 $Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$, $Co^{2+}$에 의해 활성이 회복되었다. Protease I은 pH8.0과 $50^{circ}C$에서, protease II는 pH8.0과 $60^{circ}C$에서 가장 높은 활성을 나타내었다. Protease I의 pI는 6.9, protease II의 pI는 7.1로 측정되었으며, 두 효소 모두 azocoll외에 azocasein과 casein도 가수분해하였으나 bovine serum albumin과 일산화탄소 산화효소는 가수분해하지 못하였다.
Two proteases, protease I and II, from cells of Acinetobacter sp. strain JC1 grown on nutrient broth were purified to better than 95% homogeneity in five steps using azocoll as a substrate. The final specific activities of protease I and II were 107 and $2,889{mu}g$ of azocoll hydrolyzed per min per mg of protein, respectively. Protease II contributed to about 95% of the total protease activity. The molecular weights of the native enzymes of protease I and II were determined to be 55,000 ad 44,000, respectively. Sodium dodecyl sulfate-gel electrophoresis revealed the two enzymes to be monomers. The enzymes were found to be serinetype proteases, and were present only in cells grown in complex medium. The activity of protease I was stimulated significantly by $Ca^{2+}$ and $Mg^{2+}$, but that of protease II was not. The enzymes were inhibited completely by $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Fe^{2+}$ and EDTA. EGTA showed no inhibitory effect on the two enzymes. The EDTA-inhibited protease II was reactivated by $Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$. Maximal reaction rate of protease I was observed at pH 8.0 and $50^{circ}C$, and that of protease II was found at pH 8.0 and $60^{circ}C$. The isoelectric points of protease I and II were found to be 6.9 and 7.1, respectively. Azocasein and casein were hydrolyzed by both enzymes, but bovine serum albumin and carbon monoxide dehydrogenase were not.
