Affinity Purification and Characterization of Polyphenol Oxidase from Helianthus tuberosus L.

Polyphenol oxidase PPO of Jerusalem artichoke Helianthus Tuberosus L. tubers was purified using a Sepharose-4B-L-tyrosine-p-amino benzoic acid affinity gel. Both nativeand SDS-gel electrophoresis analyses of the purified PPO gave a single band ca. 65 kDa based on SDS-PAGE , indicating that it is a monomer. The purified PPO showed activity towards diphenolic and triphenolic substrates but not with the monophenolic substrates, suggesting that it lacks monophenolase activity. The optimum temperature and pH values vary between 20-35oC and 5.0-8.0, respectively, depending on the substrate used; for catechol, the optimum temperature and pH values were found to be 20oC and 7.0, respectively. The purified enzyme was relatively stable at 40oC but unstable at higher temperatures. Furthermore, IC50 values for various inhibitors and inhibition modes were also determined using catechol as a substrate; β-mercaptoethanol showed the strongest inhibition, followed by 2-mercapto benzothiazol, glutathione, L-cysteine and dithioerythritol, respectively

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