Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Isolation and Characterization of Bacillus subtilis CH-10 Secreting Cellulase from Cattle Manure

우분으로부터 Bacillus subtilis CH-10의 분리 및 균주가 분비하는 Cellulase의 특성에 관한 연구

  • Kim, Tae-Il (National Livestock Research Institute, Rural Development Administration) ;
  • Han, Jung-Dae (National Livestock Research Institute, Rural Development Administration) ;
  • Jeon, Byoung-Soo (National Livestock Research Institute, Rural Development Administration) ;
  • Ha, Sang-Woo (National Livestock Research Institute, Rural Development Administration) ;
  • Yang, Chang-Bum (National Livestock Research Institute, Rural Development Administration) ;
  • Kim, Min-Kyun (Division of Applied and Chemistry, School of Agricultural Biotechnology center for Plant Molecular Genetics and Breeding Research, College of Agriculture and Life science, Seoul National University)
  • 김태일 (농촌진흥청 축산기술연구소) ;
  • 한정대 (농촌진흥청 축산기술연구소) ;
  • 전병수 (농촌진흥청 축산기술연구소) ;
  • 하상우 (농촌진흥청 축산기술연구소) ;
  • 양창범 (농촌진흥청 축산기술연구소) ;
  • 김민균 (서울대학교 농생대 응용생물화학부)
  • Published : 1999.01.01
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Abstract

A bacterium producing the extracellular cellulase was isolated from cattle feces and screened as cellulase activity was excellent upon congo red straining method and activity measurements. Isolate was identified as Bacillus subtilis CH-10 on the basis of morphological and biochemical properties as well as cellular fatty acids composition. The enzyme which the isolate secretes had the optimum initial pH and temperature for its induction was 7.5 and 50${\circ}C$, respectively. The maximum CMCase activity in crude enzyme solution was observed at pH 7.5 and 75${\circ}C$ and was stable for pH 7.5 to 9.0 to maintain 70% activity. When the isolate was cultured in CMC media at 37${\circ}C$ for 24 hrs, CMCase and FPase activity was 1.13 U/㎖and 0.16U/㎖, respectively whereas Avicelase and ${\beta}$-glucosidase activity was not detected. When crude supernatant was used for zymogram, three major bands, cel 1, cel 2 and cel 3, were detected approximately 39, 41 and 57 KDa, respectively on CMC-SDS-PAGE.

우분으로부터 cellulase를 생산하는 미생물을 congo red 염색과 활성측정을 통해 선발하여 cellulase 활성이 우수한 균을 분리하였다. 분리균은 생화학적, 형태학적, 균체 지방산 조성을 근거로 Bacillus subtilis CH-10으로 동정하였다. 분리균이 분비하는 효소학적 특성 중 효소생산 조건은 초기 pH7.5 및 배양돈도 50${\circ}C$ 그리고 48시간 배양이 가장 적합하였다. 조효소액에서 CMCase 최적 온도는 75${\circ}C$이었고, 온도안정성은 50${\circ}C$까지 70%의 효소활성을 유지하였다. 조효소액에서 CMCase 최적 pH는 7.5이었고, pH 안정성은 pH7.5~9.0 영역에서 70%의 효소활성을 유지하였다. 분리균을 CMC 배지에서 37${\circ}C$, 24시간 배양시 나타난 CMCase와 Fpase 활성은 각각 1.13 U/㎖와 0.16U/㎖ 였으나 avicelase와 ${\beta}$-glucosidase의 활성은 검출되지 않았다. CMC-SDS-PAGE 방법으로 3개의 효소활성 band를 확인하였고, Cel 1 및 2 그리고 Cel 3의 분자량은 각각 약 39 및 41 그리고 57kDa이었다.

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