Coconut peroxidase isoenzymes: isolation, partial purification and physicochemical properties

Abstract

Peroxidases (donor: hydrogen peroxide oxidoreductase; EC 1.11.1.7) from normal and mutant (makapuno) coconut endosperms were compared in terms of their specific activities and isoenzyme patterns, physicochemical properties and ability to oxidize IAA. Makapuno peroxidases have significantly higher activity during the early stages of development and significantly lower activity at maturity when compared with the normal. Six anodic isoenzymes were detected at pH 8.9 from both sources by polyacrylamide gel electrophoresis. Both normal and makapuno crude peroxidases have optimum activity at pH 5.5 and are thermostable over a wide range of temperature. The V max was obtained at 1 × 10 −2 M hydrogen peroxide while the apparent K m was at ca 1 × 10 −3 M hydrogen peroxide. In general, no marked difference was noted between the physicochemical properties of peroxidases from both sources. Isolation and partial purification of the isoenzymes were performed by conventional methods including ammonium sulfate fractionation, DEAE Sephadex A-50 ion exchange chromatography and Sephadex G-200 gel filtration. Peroxidase activity was localized in the 50–95% ammonium sulfate precipitate. Gel filtration resolved three protein peaks with peroxidase (Px) activity, the electrophoretic mobilities of which corresponded to Px3, Px4 and Px5. Px5 was purified 17 600-fold and found to be a tetramer with a native MW of 196000 and a subunit MW of 55000. Px3 and Px4 were monomers with MWs of 36300 and 46800, respectively. IAA oxidase activity was detected in the partially purified peroxidases from developing normal endosperms but not from mature endosperms.