Purification and Characterization of Polyphenol Oxidase From Muscat Bailey A Grape Juice

¹ The Institute of Enology and Viticulture, Yamanashi University, Kofu 400-0005, Japan
² International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.

jcacho{at}posta.unizar.es

Polyphenol oxidase of Muscat Bailey A (Muscat Hamburg x Bailey) (MBA) was purified in a homogeneous form on SDS-PAGE and some of its properties were determined. The enzyme was extracted from the precipitate of MBA juice and purified by Sephadex G-100, hydroxyapatite, DEAE-Sepharose, and Ultrahydrogel 250 (HPLC) column chromatographies. The specific activity toward catechol increased 84-fold and the recovery was about 1%. The molecular weight of the enzyme was 40 kDa (SDS-PAGE) or 42 kDa (gel filtration). It had an optimum pH of 6.3 and was stable between pH 6 and 8. The optimum temperature for activity was 25°C to 30°C and the enzyme remained stable up to 30°C. When tested for substrate specificity, the enzyme catalyzed the oxidation of o-dihydroxy phenolic compounds but showed no activity toward monohydroxy phenols. The Km value toward catechol was 18.4 mM. Catecholase activity (browning activity) was well inhibited by ascorbic acid, potassium pyrosulfite, and other inhibitors, but the enzyme remained active in the presence of 1% SDS.

Key words: polyphenol oxidase, purification

Submitted on March 16, 1998
Revised on December 15, 1998