무당벌레 소화기관으로부터 산성 단백질 분해효소를 생산하는 Staphylococcus sp. CB2-3의 분리 및 특성 = Isolation and Characterization of Acid Protease Produced by Staphylococcus sp. CB2-3 from Digestive Organ of Harmonia axyridis

국문 초록 (Abstract)

무당벌레 소화기관으로부터 단백질 분해 우수세균 6균주를 분리하였다. 단백질 분해세균의 16S rRNA 유전자 염기서열을 해석하여 계통학적 특성을 검토한 결과, Staphylococcus sciuri subsp. sciuri (3균주), Bacillus subtilis (1균주), Bacillus thuringiensis (2균주)로 확인되었다. 이들 균주 중 pH 5.0 배지에서 58.5 U/ml의 높은 효소 활성을 나타내는 Staphylococcus sp. CB2-3을 최종 선발하였다. 효소의 특성을 조사한 결과, pH 4.0-6.0에서 높은 활성을 나타내어 산성 단백질 분해효소임이 확인되었다. 효소의 최적 반응 온도는 $40^{\circ}C$ 이었으며, $30-50^{\circ}C$의 범위에서 80% 이상의 효소 활성을 유지하였다. Staphylococcus sp. CB2-3 균주의 생육과 효소 활성을 위한 최적의 배지성분을 조사하였다. 탄소원으로 0.5% 솔비톨을 첨가하였을 때 효소 활성이 2배로 증가되었으며, 질소원으로 0.5% 탈지유를 첨가한 경우 효소 활성이 2.5배 증가되는 것으로 나타났다. 무기염류로는 KCl, $K_2HPO_4$, $FeSO_4$를 첨가하였을 때 효소 활성이 가장 높은 반면에 2가 금속이온인 $Co^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Cu^{2+}$를 첨가하였을 때는 균의 성장과 효소 활성이 심하게 저해되는 것으로 나타났다.

다국어 초록 (Multilingual Abstract)

Six protein-degrading bacteria were isolated from digestive organ of Harmonia axyridis. These isolates were categorized as Staphylococcus sciuri subsp. sciuri (3 strains), Bacillus subtilis (1 strain), and Bacillus thuringiensis (2 strains) by 16S rRNA gene sequence analysis. The Staphylococcus sp. CB2-3 was selected as a protease-producing bacterium which showed the highest protease activity of 58.5 U/ml at the pH 5.0 medium. The optimal pH and temperature of protease activity were pH 5.0 and $40^{\circ}C$, respectively. This acid protease had a relatively high stability of 80% between $30-50^{\circ}C$ at broad temperature range. The opimal medium compositions of carbon, nitrogen and mineral source for cell growth and protease activity were investigated. When sorbitol (0.5%) was used as carbon source, enzyme activity was increased about 2 times than that of the basal medium. When skim milk (0.5%) was used as nitrogen source, activity was increased about 2.5 times than that of the control. Cell growth and enzyme activity were increased by mineral source such as KCl, $K_2HPO_4$, $FeSO_4$, but was completely inhibited by divalent ions such as $Co^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Cu^{2+}$.

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