Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Characterization of Enzymatic Properties of Streptomyces albus Amylase Expressed in Recombinant Bacillus subtilis

재조합 Bacillus subtilis 에서 발현된 Streptomyces albus 유래 amylase 의 효소공학적 특성

  • Park, Geun-Woo (Department of Food Science and technology and Research Center for New Bio-materials in Agriculture, Seoul National University) ;
  • Kim, Myoung-Dong (Department of Food Science and technology and Research Center for New Bio-materials in Agriculture, Seoul National University) ;
  • Ahn, Jang-Woo (Department of Food Science and Technology, Chungkang College of Cultural Industries) ;
  • Kim, Young-Bae (Department of Food Science & Technology, Korea University) ;
  • Seo, Jin-Ho (Department of Food Science and technology and Research Center for New Bio-materials in Agriculture, Seoul National University)
  • 박근우 (서울대학교 식품공학과 및 농업생물신소재 연구센터) ;
  • 김명동 (서울대학교 식품공학과 및 농업생물신소재 연구센터) ;
  • 안장우 (청강문화산업전문대학 식품공업과) ;
  • 김영배 (고려대학교 식품공학과) ;
  • 서진호 (서울대학교 식품공학과 및 농업생물신소재 연구센터)
  • Published : 1998.12.01
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Abstract

The research was undertaken to characterize enzymatic properties of Streptomyces albus amylase expressed in recombinant Bacillus subtilis. Molecular weight and pI of the purified enzyme were estimated to be 50 kD by SDS-PAGE and 4.3 by isoelectric focusing. The optimum temperature and optimum pH were $45^{\circ}C$ and 6.0, respectively. D-and Z-value were estimated to measure thermostability of the purified enzyme. The Z-value was estimated $17.7^{\circ}C$, which is lower than typical amylase. Maltotetraose was produced as a major component from soluble starch in the early state of reaction but gradually degraded to maltose. Thin layer chromatography was also performed to analyze the reaction products. The parameters involved in Michaelis-Menten enzyme kinetics were found to be the maximum velocity of 0.37 mM/min and the Michaelis constant of 0.13%, respectively.

본 연구에서는 재조합 Bacillus subtilis에서 발현된 Streptomyces albus KSM-35유래의 amylase를 정제하고 특성을 구명하였다. 정제된 효소는 SDS-PAGE를 통하여 분자량이 약 50 kD인 것으로 밝혀졌으며, isoelectric focusing을 통하여 측정된 pI값은 약 4.3이었다. 효소의 최적 반응온도는 $45^{\circ}C$이었으며 최적의 pH는 6.0이었다. D-value는 45, $55^{\circ}C$에서 각각 279분, 191분이었고 D-value로부터 계산된 Z-value는 $17.7^{\circ}C$였다. 수용성 전분용액을 기질로 사용한 효소반응의 초기에는 maltotriose, maltopentaose와 maltotetraose가 주로 생성되었지만 시간이 경과함에 따라 이들의 농도는 감소하였고 maltose의 농도가 점차 증가하였다. 이러한 반응 생성물의 분해는 Thin layer chromatography를 통하여 확인할 수 있었다. 기질에 의한 저해가 없다고 가정하고 Michaelis-Menten kinetics를 이용하여 속도상수를 추정하였을 때 최대 반응속도는 0.37 mM/min, Michaelis-Menten 상수는 0.13% (w/v)로 나타났다.

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