Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Biochemical characterization of a novel extracellular pullulan 6-glucanohydrolase from Bacillus circulans S-1

  • Lee, Moon-Jo (Department of Food Technology, Dongeui Institute of Technology, Pusan) ;
  • Park, Cheon (Department of Biochemsitry and Molecular Biology, College of Oriental Medicine, Dongguk University) ;
  • Park, Joon-Ho (Department of Biochemsitry and Molecular Biology, College of Oriental Medicine, Dongguk University) ;
  • Chung, Kang-Hyun (Department of Food Science and Technology, Seoul National University of Technology) ;
  • Nam, Kyung-Soo (Department of Pharmacology, College of Medicine, Donggut University) ;
  • Park, Jin-Woo (Department of Food Technology, Dongeui Institute of Technology, Pusan) ;
  • Kim, Cheorl-Ho (Department of Biochemsitry and Molecular Biology, College of Oriental Medicine, Dongguk University)
  • Published : 2000.06.01
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Abstract

Bacillus circulans S-1 extracellular pullulan 6-glucanohydrolase (EP) (EC 3.2.1.41) has been characterized with a purified enzyme of 140 kDa. The N-terminal amino acid sequence of the purified enzyme was P-L-N-M-S-Q-P. The enzyme displayed a temperature optimum of around $60^{\circ}C$ and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at $4^{\circ}C$ for 48h. The presence of substrate allowed the protection of the enzyme from heat inactivation. The activity of the enzyme was stimulated by several metal ions such as Mn2+ and Ca2+. The enzyme had an apparent Km of 7.92 mg/ml for pullulan. The purfied enzyme completely hydrolysed pullulan to maltotriose.

Bacillus circualans S-1으로부터 새로운 균체외 pullulan 6-glucanohydrolase(EP)를 정제하였다. 정 제된 EP는 SDS하에서 140kDa의 분자량을 나타내었으며, pI는 5.5이었다. SDS-PAGE에 의해 분석된 이 EP는 Schiff staining에 대하여 negative이었으며, 또한 아미노 말단기 순서는 P-L-N-M-S-Q-P이었 다. 정제된 EP는 6$0^{\circ}C$ 부근의 최적온도와 pH 9.0 부근에서 최적 pH를 나타내었으며, pH 4.0에서 pH 11 까지 4$^{\circ}C$에서 24시간동안 반응에서도 안정하였다. 또한 기질로 사용된 Pullulan은 열불안정화로부터 효 소를 보호하였으며, 그 범위는 기질 농도에 의존하였다. 이 EP의 활성은 Mn2+, Ca2+ 이온 에 의하여 활성화되었으며, amylopectin, glycogen, $\alpha$,$\beta$-limited dextrin 및 pullulan의 $\alpha$-1,6-linkage를 가수분해 하였다. 정제된 EP는 pH 9.0 및 5$0^{\circ}C$에서 측정하였을 때 pullulan의 경우는 7.92mg/ml의 Km값을 각각 나타내었다. 또한 정제된 EP는 pullulan을 maltotriose까지 완전히 가수분해하였다. 그리고 Mouse anti-serum과 함께 western blotting 분석결과 정제된 EP는 배양과정중 단일 형태로 생산되는 것으로 나타났다.

Keywords

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