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Journal of the Korean Chemical Society (JKCS)

ISSN 1017-2548(Print)
ISSN 2234-8530(Online)
Volume 24, Number 1
JKCSEZ 24(1)
February 20, 1980 

 
Title
Studies on Pyrocatechase from a Soil Bacterium (Ⅰ). Purification and Characterization of Pyrocatechase

토양 박테리아로부터의 Pyrocatechase 에 관한 연구 (제1보). 효소정제와 특성연구
Author
Yoen-Bo Chung, Hyun-Jae Lee

정연보, 이현재
Keywords
Abstract
토양에서 분리한 Pseudomonadaceae 속 박테리아로부터 pyrocatechase를 추출, 분리 정제하였으며, 이 효소의 특성을 검토한 결과 pyrocatechase는 catechol에 대하여 기질 특이성을 보여줌을 알았다. 효소 활성도의 최적조건은 pH 7∼10 부근과 온도 35℃임을 알았으며, catechol에 대한 Km값은 1.9 × 10-6M 로 얻어졌다. 기질 유도체에 의한 효소 저해 실험결과 벤젠 고리의 ortho 위치에 두개의 수산기는 효소-기질간의 결합반응에 참여될 것이라고 추측했다. 기타 SH-잔기와 작용하는 화합물 또는 중금속 이온등의 첨가에 따른 효소 활성도의 저해 효과를 검토 하였으며, 효소 활성부위에 대하여도 검토해 보았다.

Pyrocatechase as a phenolytic dioxygenase was extracted from the benzoate-induced cells of a soil bacterium, a member of Pseudomonadaceae, and purified partially by DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel filtration. Final preparation of the enzyme yielding 200 fold purification over the crude extracts showed a specific activity of about 40 μmoles per minute per mg protein based on catechol as the substrate. The enzyme showed a very limited substrate specificity towards catechol for its catalytic activity. Based on the inhibition study with the substrate analogues, it was assumed that ortho dihydroxy groups on the aromatic ring may participate in the enzyme-substrate binding. The Km value for catechol was obtained as 1.9 × 10-6 M, and the optimum activity of the enzyme was obtained at the pH range of 7∼10 and 35℃. With SH-group blocking agents the enzyme was inhibited seriously.The activity of enzyme was also inhibited by the addition of some heavy metals, Ag+ and Cu2+, but was not affected by EDTA. General property of the enzyme was characterized and the possible nature of the enzyme active center was also discussed.

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