Purification and Some Properties of Polyphenol Oxidase from Arrowroot

칡 뿌리의 Polyphenol Oxidase의 정제 및 성질에 관한 연구

Acetone powder was prepared from raw arrowroots and the polyphenol oxidases of crude enzyme prepared from acetone powder were identified 5 isoenzymes by staining with catechol containing 0.05% phenylene diamine. The crude enzyme was passed through the columns of ion exchangers and gel permeation to fractionate the polyphenol oxidases. The main fraction of polyphenol oxidase appeared to be purified by 94-fold, with the activity yield of 45.4%, and its molecular weight was determined as 38,500 by poly acrylamide gel electrophoresis. The optimal pH and temperature for the enzyme activity were pH 7.5 and $50^{\circ}C$, respectively. The purified enzyme showed a high affinity for catechol and pyrogallol. The Michaelis constant for catechol was calculated to be 16.67mM according to the Lineweaver-Burk method. The enzyme activity was strongly inhibited by L-ascorbic acid, sodium bisulfite, EDTA and KCN, and totally inhibited, by $Fe^{3+}$ at a concentration of 1mM. However the enzyme was activated by $Zn^{2+}$ approximately 1.7 times at the same concentration.

칡뿌리로 부터 칡뿌리 acetone분말을 제조하고 polyphenol oxidase를 추출, 정제하여 정제효소의 효소학적 특성을 검토하였다. 조효소는 DEAE-Cellulose, DEAE-Sephadex A-50, Sephadex G-100 column chromatography에 의하여 정제되었으며, 정제효소의 비활성은 94배, 정제수율은 45.4%이었다. 정제효소는 catechol 및 pyrogallol에 대하여 감한 친화성을 나타내었으며, km 값은 catechel을 기질로 하였을 때 16.67mM이었다. 작용 최적 pH는 7.5, 최적온도는 $50^{\circ}C$에서 가장 잘 작용하였고 1mM L-ascorbic acid, sodium bisulfite, EDTA, KCN 및 $Fe^{3+}$ 이온에 의하여 심한 저해를 나타내었으며, $Zn^{2+}$ 이온을 약 1.7배 정도의 활성을 증가시켰다. 조효소액을 전기영동하여 catechol로 활성염색 하였을 때 5개 isoenzyme이 확인되었으며 그 중 분자량 35,000의 것이 가장 강한 활성을 나타내었다.