Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Purification and Characterization of Protease from Entomopathogenic Fungus Beauveria bassiana

곤충 병원성 곰팡이 Beauveria bassiana로부터 Protease의 정제와 특성

  • Ko, Hwi-Jin (Department of Genetic Engineering, Sung Kyun University) ;
  • Kim, Hyun-Kyu (Department of Genetic Engineering, Sung Kyun University) ;
  • Kim, Beom-Gi (Department of Genetic Engineering, Sung Kyun University) ;
  • Kang, Sun-Chul (Department of Biotechnology, Taegu University) ;
  • Kwon, Suk-Tae (Department of Genetic Engineering, Sung Kyun University)
  • 고휘진 (성균관대학교 유전공학과) ;
  • 김현규 (성균관대학교 유전공학과) ;
  • 김범기 (성균관대학교 유전공학과) ;
  • 강선철 (대구대학교 생물공학과) ;
  • 권석태 (성균관대학교 유전공학과)
  • Published : 1997.10.31
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Abstract

Extracellular protease (bassiasin I), from the culture filtrate of entomopathogenic fungus Beauveria bassiana ATCC7159, was successively purified by precipitation with ammonium sulfate followed by DEAE-Sephadex A-50, CM-cellulose and Hydroxyapatite column chromatography. A typical procedure provided 41-fold purification with 13.6% yield. The molecular weight of the purified pretense (bassiasin I) was found to be approximately 32,000 by SDS-PAGE. Isoelectric-focusing analysis of the enzyme showed a pI of 9.5. $NH_2-terminal$ sequence of the pretense showed homology with those of the fungal proteases. The enzyme has an optimal pH for activity at 10.5 and is stable over pH 5.0-11.0. The maximum activity of the enzyme was at $60-65^{\circ}C$, and approximately 20% activity remained at $60^{\circ}C$ after 120 min. The pretense was inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DIPF).

곤충 병원성 곰팡이 Beauveria bassiana ATC7159의 배양여액으로부터 균체외 protease (basiasin I)를 Ammonium sulfate 침전, DEAE-Sephadex A-50, CM-cellulose 및 Hydroxyapatite column chromatography를 수행하여 완전히 정제하였다. 이 과정으로 41배 정제되었으며 정제수율은 13.6%였다. 정제된 bassiasin I의 분자량은 SDS-PAGE 상에서 약 32,000 Da이며 pI값은 9.5로 확인되었다. $NH_2$ 말단 아미노산 서열은 다른 곤충 병원성 곰팡이가 생산하는 pretense들과 높은 상동성을 보였다. Protease 활성의 최적 pH는 10.5 부근이며, pH 5.0-11.0범위에서 안정하였다. 최대 활성온도는 $60-65^{\circ}C$이며, $60^{\circ}C$에서 120분후에 약 20%의 잔존활성을 보였다. 이 pretense는 phenylmethylsulfonyl fluoride (PMSF) 및 diisopropyl fluorophosphate (DFP)에 의해 저해된다.

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