Characterization of an Alkaline Protease from an Alkalophilic Bacillus pseudofirmus HS-54

호알칼리성 Bacillus pseudofirmus HS-54가 생산하는 알칼리성 Protease의 특성

An alkalophilic bacterium producing alkaline protease was isolated from waste water and solar saltern sample and identified as Bacillus pseudofirmus HS-54 based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The HS-54 protease was purified to homogeneity using ammonium sulfate precipitation, DEAE cellulose column chromatography, and sephadex G-100 gel filtration with a 4.0 purification fold. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 27 kDa. The optimal pH and temperature for the purified protease activity were 10.0 and $50^{\circ}C$, respectively. The purified enzyme was relatively stable at the pH range of 6.0-11.0 and at the temperature below $50^{\circ}C$. This enzyme was activated by $Ca^{2+}$ and $Mg^{2+}$ and inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Al^{3+}$, $Ag^{2+}$. And this enzyme was strongly inhibited by PMSF, suggesting that it belongs to the serine protease superfamily.

알칼리성 protease를 생산하는 호알칼리성 균주를 분리하여 Bacillus pseudofirmus HS-54로 동정하였고, HS-54가 생산하는 알칼리성 protease를 ammonium sulfate 침전, DEAE cellulose chromatography, sephadex G-100 gel filtration을 통과시켜 정제하였는데, 정제된 protease의 분자량은 27 kDa이었다. 정제된 효소의 반응최적 pH는 10.0이었고 pH 7.0-11.0에서 비교적 안정하였다. 또한 정제된 효소의 반응최적 온도는 $50^{\circ}C$이었고 $10-55^{\circ}C$에서 안정하였다. 금속이온에 대한 영향은 $Ca^{2+}$와 $Mg^{2+}$ 등에 의해 효소활성이 촉진되었으나, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Al^{3+}$ 등에 의해서 효소활성이 저해되었다. 본 효소는 PMSF에 의해 강하게 저해를 받는 것으로 보아 serine protease에 속하는 것으로 판단된다.