Isolation and purification of polyphenol oxidase from a new variety of potato

Abstract

Polyphenol oxidase (E.C. 1.10.3.1) was extracted from potato ( Solanum tuberosum var. Tebere) tissue and purified using the techniques of ammonium sulphate fractionation, DEAE-cellulose chromatography and filtration through Sephadex G-200. Four isoenzymes with different relative mobilities were detected by polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 112 000 as estimated by gel filtration on Sephadex G-200 and an apparent Km of 8·68 × 10 −5 m for chlorogenic acid as substrate, and pH optimum of 6·6. The heat inactivation plot at 65°C at different pH values was biphasic. It was also found that the Arrhenius plot for chlorogenic acid oxidation was biphasic showing a discontinuity at 8°C with an increased slope as temperatures were reduced to 1°C. Cinnamic and ferulic acids were competitive inhibitors with respect to chlorogenic acid with Ki values of 2 × 10 −5 and 5 × 10 −6 m , respectively.