본 연구는 부산 인근의 해수욕장에서 얻은 해수에서 protease를 생산하는 균주를 분리하여 동정하고 균주의 배양학적인 특성과 protease의 효소학적 특성을 확인하였다. 해수에서 분리한 protease를 생산하는 미생물은 16S rDNA sequencing을 통해 Micrococcus sp. PS-1으로 동정하였다. Protease 생산의 최적조건은 2% skim milk와 1% NaCl이 포함된 pH 7.0의 LB배지에 48시간 배양이었다. 효소의 부분정제를 위해 ultrafiltration과 acetone 침전법을 사용하였고, zymography를 통해 분자량이 35.0 kDa과 37.5 kDa인 protease를 확인하였다. 또한 효소의 최적 활성은 pH 9.0와 $37^{circ}C$에서 나타났고, 효소는 pH 8.0에서 11.0까지, $25^{circ}C$에서 $37^{circ}C$까지 80% 이상의 효소활성이 유지되어 안정한 것으로 확인되었으며 PMSF, EDTA 처리시 protease가 저해되는 것을 통해 alkaline metallo-serine protease로 확인되었다.
The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria-Bertani (LB) medium. Its optimal temperature and pH for growth were $30^{circ}C$ and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and $37^{circ}C$, and its activity was stable at pH 8.0-11.0 and $25-37^{circ}C$. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.
