국내 토양균 Actinomycestes CS624가 분비하는 protease의 특성연구

Abstract

Actinomycetes CS624, isolated from Korean soil, showed 100% 16S rRNA gene sequence homology with Streptomyces murinus NBRC 12799(T), Streptomyces costaricanus NBRC 100773(T), Streptomyces griseofuscus NBRC 12870(T), and Streptomyces graminearus NBRC 15420(T). It produced neutral extra cellular proteases, when cultured in a medium containing 1.5% glucose, 1.5% oat meal, 0.45% K2HPO, and 0.45% NaHPO₄at 28℃ and 180rpm. Three forms of protease, designated as S-2, D-1 and D-2, were purified through Sepherose CL-6B column chromatography followed by DEAE- Sepherose CL-6B ion exchange column chromatography. The purified enzymes were then analyzed by using 10% SDS-PAGE, which revealed that the molecular masses of the purified enzyme, S-2, D-1 and D-2, were about 20, 30 and 50 kDa, respectively. The temperature optima for S-2, D-1 and D-2 were 70, 50 and 50℃, respectively. The pH optima for S-2, D-1 and D-2 were pH6, pH7 and pH6, respectively. Intact enzyme activity remained in existence when S-2, D-1 and D-2 were treated for up to 2h at 70℃, 50℃ and 40℃, respectively. Moreover, all the proteases were stable in the pH range of 5 to 8. The activity of D-1 and D-2 were completely inhibited by Fe2+ and Mn2+ whereas S-2 activity was suppressed only by Fe2+. Furthermore, EDTA inhibited the activity of D-1 and D-2, but not that of S-2. In contrast, PMSF did not affect the activity of all form of proteases.