Purification and Characterization of Fibrinolytic Enzyme Produced by Bacillus subtilis K7 Isolated from Korean Traditional Soy Sauce

한국재래간장 발효균 Bacillus subtilis K7 유래의 혈전용해 Protease의 정제 및 특성

  • Kim, Doo-Young (Department of Applied Microbiology, College of Natural Resources, Yeungnam University) ;
  • Lee, Eun-Tag (Department of Applied Microbiology, College of Natural Resources, Yeungnam University) ;
  • Kim, Sang-Dal (Department of Applied Microbiology, College of Natural Resources, Yeungnam University)
  • 김두영 (영남대학교 자연자원대학 응용미생물학과) ;
  • 이은탁 (영남대학교 자연자원대학 응용미생물학과) ;
  • 김상달 (영남대학교 자연자원대학 응용미생물학과)
  • Published : 2003.08.30

Abstract

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

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