Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Production and Characterization of Alkaline Protease of Micrococcus sp. PS-1 Isolated from Seawater

해수에서 분리한 Micrococcus sp. PS-1이 생산하는 단백질 분해효소의 생산과 효소학적 특성

  • Jin, Young-Rang (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Yu, Sun-Nyoung (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Kwang-Youn (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Sang-Hun (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Park, Seul-Ki (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Hyeun-Kyeung (Medical Research Institute, Pusan National University) ;
  • Lee, Yong-Seok (Department of Biotechnology, Dong-A University) ;
  • Choi, Yong-Lark (Department of Biotechnology, Dong-A University) ;
  • Ji, Jae Hoon (Genome Instability Research Center, Ajou University School of Medicine) ;
  • Ahn, Soon-Cheol (Department of Microbiology and Immunology, Pusan National University School of Medicine)
  • 진영랑 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 유선녕 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김광연 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김상헌 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 박슬기 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김현경 (부산대학교 의학연구원) ;
  • 이용석 (동아대학교 생명과학자원대학 생물공학과) ;
  • 최용락 (동아대학교 생명과학자원대학 생물공학과) ;
  • 지재훈 (아주대학교 의과대학 유전체불안정제어 연구센터) ;
  • 안순철 (부산대학교 의학전문대학원 미생물학 및 면역학 교실)
  • Received : 2013.01.18
  • Accepted : 2013.02.20
  • Published : 2013.02.28
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Abstract

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.

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.

Keywords

References

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