Abstract
A crystalline alkaline pretense- producing Streptomyce sp. YSA-130 was isolated from soil in alkaline medium(pH 10.5). The optimum culture condition of Streptomyce sp. YSA-130 for the production of alkaline protease was as follows; 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% Na$_2$CO$_3$, pH 10.5, 3$0^{\circ}C$, and 12 hr. The alkaline pretense from the culture broth of Streptomyce sp. YSA-130 was purified about 24 folds by ammonium sulfate precipitation , dialysis, DEAE-cellulose ion exchange chromatography, gel filtration on Sephadex G-15 and crystallization. Optimum temperature and pH of purified enzyme were 6$0^{\circ}C$, and 11.5. Temperature and pH stability of purified enzyme were 5$0^{\circ}C$, and 5.5-12.0. Calcium ion was effective to stabilize the enzyme at higher temperature. The molecular weight of the purified enzyme was approximately 30,000. The purified enzyme was inactivated by diisopropyl flurophosphate(DFP) but not affected by metal ion, EDTA, sulfhydryl reagent and stable detergent.
토양으로부터 분리한 Streptomyces sp. YSA-130으로부터 활성이 좋은 결정화된 alkaline protease를 분리하였다. Alkaline protease 생산의 최적 배양조건은 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% $Na_2$CO$_3$ 3$0^{\circ}C$, pH 10.5에서 72 시간 배양하였을 때 였다. Alkaline protease이 정제는 (NH$_4$)$_2$SO$_4$. 분별침전, 투석, DEAE cellulose column chromatography, Sephadex G-75 gel filtration, crystallization으로 하였으며, 그 결과 비활성도 14,290unit/mg, 정제도 23.8 배였고, 수율은 20.0% 이었다. Alkaline protease의 반응 최적온도와 pH는 6$0^{\circ}C$ 와 11.5이었으며, 효소의 pH 안정성은 5.5-12.0에서 안정하였고, 온도 안정성은 5$0^{\circ}C$까지 안정하였으며, $Ca^{++}$ ion 첨가시 6$0^{\circ}C$까지 안정성이 증가하였다. Alkaline protease의 분자량은 30,000이었으며 금속이온, EDTA, 환원제는 활성에 영향이 없었고 DFP에 의해 저해되었다. 계면활성제에 저항성이 크고 $H_2O$$_2$에 대한 잔존활성은 60% 을 유지하였다.