Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Purification and Characterization of Collagenase Produced by Staphylococcus aureus JJ-11 Isolated from the Human Skin

피부에서 분리한 Staphylococcus aureus JJ-11이 생산하는 collagenase의 정제 및 특성

  • Lee Jin-Kyoung (Division of Biological Sciences, Pusan National University) ;
  • Kim Hae-Nam (Department of Beauty Care, Masan College) ;
  • Kang Ho-Young (Division of Biological Sciences, Pusan National University) ;
  • Jun Hong-Ki (Division of Biological Sciences, Pusan National University)
  • 이진경 (부산대학교 자연과학대학 생명과학부) ;
  • 김해남 (마산대학 뷰티케어과) ;
  • 강호영 (부산대학교 자연과학대학 생명과학부) ;
  • 전홍기 (부산대학교 자연과학대학 생명과학부)
  • Published : 2006.04.01
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Abstract

A bacterial strain, identified as Staphylococcus aureus JJ-11, producing collagenase was isolated out of 40 persons having skin troubles. S. aureus JJ-11 produced collagenase optimally in the media containing 1.5%(w/v) gelatin, 1%(w/v) yeast extract, 0.4%(w/v) $K_2HPO_4$, 0.005%(w/v) $NiSO_4{\cdot}6H_2O$ at $37^{\circ}C$ for 18 hrs. The collagenase produced by Staphylococcus aureus JJ-11 was purified at 6.66-folds purity through application of chromatography with Amberlite IRA-900 and Sephacryl S-300 HR columns. The molecular weight of the partially purified enzyme was estimated to be 62 kDa by SDS-PAGE. The protein exhibited optimum enzymatic activity at pH 7.0, and showed a stable activity at pH 4-8. The optimum temperature for collagenase was at $37^{\circ}C$, and activity was maintained upto $40^{\circ}C$. The enzyme activity was slightly elevated in the presence of divalents such as, $Fe^{2+},\;Co^{2+}\;and\;Ba^{2+}$ However, the activity was inhibited in the presence of $Sr^{2+}\;or\;Hg^{2+}$. The inhibition of activity by O-phenanthroline and EDTA suggested that the enzyme may contain metal which is required for activity. The enzyme showed the highest activity when insoluble collagen (type I) was, used as a substrate.

피부 트러블을 가진 남,여 40명의 피부에서 분리한 collagenase를 생산하는 균주를 분리, 동정한 결과 Staphylococcus aureus로 판명되었으며 이를 S. aureus JJ-11이라 명명하였다. S. aurells JJ-11 균주의 collagenase의 최적 생산 조건은 1.5%(w/v) gelatin, 1%(w/v) yeast extract, 0.4% (w/v) $K_2HPO_4$, 0.005%(w/v) $NiSO_4{\cdot}6H_2O$를 함유한 배지 (pH 7.0)에서 $37^{\circ}C$, 200 rpm으로 18시간 동안 배양하는 것이다. 분리 균주가 생산하는 collagenase를 정제하기 위해서 amberlite IRA-900과 sephacryl S-300 HR columns를 이용하였고, 6.66-folds로 정제되었다. S. allreus JJ-11 균주가 생산하는 collagenase를 정제한 결과 분자량은 약 62 kDa이었으며, pH 7.0과 $37^{\circ}C$에서 각각 최대의 활성을 가졌고, pH와 온도에 대한 안정성은 pH 4.0-8.0, $40^{\circ}C$까지 100%의 활성이 있었다. 금속이온에 대해서는 $Fe^{2+},\;Co^{2+},\;Ba^{2+}$ 존재 하에서는 5 mM 농도에서도 활성을 유지하였다. $Sr^{2+},\;Hg^{2+}$에서는 30% 이상이 저해를 받는 것으로 확인되었다. 또한 EDTA와 O-phenanthroline에 의해 65% 이상이 저해되는 일 반적인 collagenase의 특정인 metalloproteinase의 특정을 보였으며, 그리고, 여러 가지 기절에 대해 효소활성을 비교한 결과, insoluble collagen (type I)에 대해 효소 활성이 가장 높았다.

Keywords

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