Purification of a Latent Form of Polyphenoloxidase from La France Pear Fruit and Its Pressure-activation

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A latent form, mostly soluble, of polyphenoloxidase of La France pear fruit (Pyrus communis) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography with DEAE-Sephadex A-25 and then DEAE-Toyopearl 650M, followed by gel permeation chromatography with Toyopearl HW-55s. The addition of 10% glycerol to the eluting buffer was needed for purification. The purified latent enzyme seemed to be a monomeric protein; the molecular weight was estimated to be 70,000 by gel permeation chromatography and 65,000 by SDS–polyacrylamide gel electrophoresis. The enzyme was activated by pressurization at 400 MPa or higher or by treatment with SDS. The highest activity was obtained by pressurization at 600 MPa and 20°C for 6 h.