Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Comparison of enzyme activities of the native and N-terminal 6xHis-tagged Fe supreoxide dismutase from Streptomyces subrutilus P5

Streptomyces subrutilus P5의 천연 Fe superoxide dismutase와 N-말단 6xHis-태그가 결합된 Fe superoxide dismutase의 활성비교

  • Received : 2016.05.20
  • Accepted : 2016.06.17
  • Published : 2016.06.30
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Abstract

This study was carried out to analyze the differences in enzyme activity and stability between the native Fe superoxide dismutase (FeSOD) and the 6xHis-tagged superoxide dismutase (6xHis-FeSOD) of Streptomyces subrutilus P5. The optimum pHs for both native FeSOD and 6xHis-FeSOD were 7, while the pH range of the activity was narrower for the 6xHis-FeSOD. The native FeSOD was stable at pH 4-9, but the 6xHis-FeSOD lost its stability at pH > 9. The temperatures of the optimum activities were same for both types of enzymes. However, the heat stability of the 6xHis-FeSOD was clearly decreased; even at $20^{\circ}C$ the enzyme lost the activity after 360 min. In contrast, the native FeSOD was stable after 720 min at below $40^{\circ}C$. $H_2O_2$ inhibition was occurred already at 0.5 mM for the 6xHis-tagged enzyme. Therefore, from the results that the 6xHis-FeSOD retained the enzyme activity at pH 6-7 and $20-40^{\circ}C$, it can be assumed that the protein structure became destabilized under different storage conditions and sensitive to the enzyme inhibitor.

본 연구는 Streptomyces subrutilus P5의 천연 Fe superoxide dismutase (FeSOD)와 유전자 재조합 기술로 생산된 6xHis-태그가 결합된 Fe superoxide dismutase (6xHis- FeSOD)의 활성을 비교하여 6xHis-태그의 효소에 대한 영향을 알아보기 위하여 수행되었다. 두 효소 모두 최적 pH는 7로 동일하였으나 6xHis-태그에 의해서 pH 범위는 축소되었다. 천연 효소는 pH 4-9의 범위에서 안정성을 보인 반면 6xHis-FeSOD는 pH 9에서 안정성이 상실되었다. 두 효소의 최적 온도는 차이가 없으나 열 안정성에 있어서는 천연 효소는 $40^{\circ}C$ 이하에서 720분까지 안정성을 유지하였으나 6xHis-FeSOD는 $20^{\circ}C$에서도 360분 이내에 활성을 잃는 것으로 나타났다. $H_2O_2$의 6xHis-FeSOD에 대한 저해는 0.5 mM에서 나타났다. 따라서 6xHis-FeSOD는 효소활성은 유지되더라도 열 안정성이 크게 감소되는 결과를 얻었다. 이것은 6xHis-태그가 활성부위 보다는 단백질 전체 구조에 더 많은 영향을 미친 결과라고 생각되었다.

Keywords

References

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