Partial Purification of a Banana Polyphenol Oxidase Using Triton X-114 and PEG 8000 for Removal of Polyphenols

Abstract

Banana pulp polyphenol oxidase was partially purified in a latent form using sequential aqueous two-phase systems based on Triton X-114 and PEG 8000/phosphate. The purification achieved from a crude extract of banana pulp was 5-fold, with 50% recovery of the activity. The (poly)phenols, including tannins, were also reduced to 6% of the original, avoiding the postpurification tanning of the enzyme and rendering only one enzymatic form in contrast to the many previously shown for this enzyme in both electrophoretic and isoelectrofocusing methods. The enzyme was kinetically characterized with its natural substrate (dopamine) and tert-butylcatechol in the presence and in the absence of the main activating agent, sodium dodecyl sulfate. In addition, the effect of several inhibitors was also tested, and the Ksi values of the two most effective substrate analogues (tropolone and kojic acid) were determined. Keywords: Aqueous two-phase system; polyphenol oxidase; banana; Triton X-114; polyethylene glycol; latent ...