Polyphenol oxidase from artichoke (Cynara scolymus L.)

Polyphenol oxidase (PPO) was purified from hearts of artichoke. Starting with a crude 20mm acetate buffer pH 5.7 extract obtained from an acetone powder originating from the plant material, the enzyme was purified 65-fold (overall yield 9%) by means of hydrophobic followed by gel filtration chromatography. The purified artichoke polyphenol oxidase (APPO), which migrated as a single band during gel filtration chromatography and chromatofocusing, showed an isoelectric point of 4.5 and a molecular weight of 116,000 dalton. The best substrates for the enzyme at pH 6.0 were 5-o-caffeoylquinic acid (5-o-CQA, according to IUPAC 1976 nomenclature) (relative activity 100%) and caffeic acid (relative activity 69%). Pyrocatechol, which was also oxidized by APPO (relative activity 42%), proved to be a less suitable substrate, whereas 1,5-dicaffeoylquinic acid and catechin were only very poorly oxidized. With each of the main substrates the optimum pH was widespread between pH 5.0 and 7.0 for 5-o-CQA and pyrocatechol, while for caffeic acid the maximum activity was widely extended between pH 6.5 and 8.0. The kinetic constants of the enzyme determined at pH 7.0 and 30 degrees C with both 5-o-CQA and caffeic acid as substrate proved to be quite similar (Km = 4.2 +/- 0.5 mm, Vmax = 135.6 +/- 14.7 U/mg and Km = 5.3 +/- 0.3 mm, Vmax = 115.1 +/- 6.1 U/mg, respectively). The activation energy of the enzyme with caffeic acid was 21.7 +/- 0.2 kJ/mole at pH 6.5. Cu++ and Fe+++ proved to activate APPO-5-o-CQA oxidation. However, APPO activation by these ions does not appear to be important enough to ascribe them a role in the enzymatic browning of stored artichoke heads. Although both ascorbic and citric acids are known to considerably improve the shelf life of artichoke heads, only ascorbic acid proved to be significantly inhibitory towards APPO.