Polyphenol Oxidase from Grapes: Precipitation, Re-Solubilization and Characterization

¹ Institute of Enology and Viticulture, Yamanashi University, Kofu, Yamanashi 400, Japan
² Department of Viticulture and Enology, University of California, Davis 95616-5270.

Samples of Koshu grape musts were prepared by pressing at different pressures, giving three different yields. The polyphenol oxidase (PPO) activity of the musts increased as the yield increased. The percentages of the protein and the total PPO activity remaining in clarified juice averaged only 11.9% and 5.8%, respectively, of their values before clarification. The insoluble precipitate, however, was enzymatically active, retaining 74.0% of the protein and 42.2% of the PPO activity when resuspended. The washed precipitate was 22.6% protein and 9.7% phenol (dry wt). The precipitate had high PPO activity towards p-cresol, (+)-catechin, (-)-epicatechin, caffeic acid, catechol, and pyrogallol. Various methods, including pH change and detergents, were tested to solubilize PPO activity from the precipitate; alkaline treatment proved most efficient. The PPO solubilized at pH 10 exhibited optimum activity at pH 6.0 and behaved as a typical PPO. Potassium cyanide inactivated the enzyme, but addition of copper sulfate was able to restore ca 90% of the original activity. The re-solubilized material was chromatographed on Sephadex G-100 to give three active fractions. These were different in pattern on disc gel electrophoresis and elution volume on gel filtration, but similar in amino acid composition. One was only partially precipitated by grape tannin under conditions that completely precipitated the other two. The second was ca 50% as active as a precipitate as it was in solution, while the third was essentially equally active in both forms. Insoluble tannin-PPO complexes formed were again re-solubilized in pH 10 buffer. About 70% of their activity and 85% of their protein could be recovered. The results show the importance of removing insolubles from must early to minimize PPO effects because insoluble but suspended precipitates are active. Re-solubilization appears unlikely in wine, but recovery of soluble PPO by alkaline treatment of precipitated PPO from must offers promise for enzyme purification.

Key words: enzymic browning, must, polyphenol oxidase, precipitation

Submitted on October 14, 1987

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