PARTIAL-PURIFICATION AND CHARACTERIZATION OF ENDIVE (CICHORIUM-ENDIVIA L) POLYPHENOLOXIDASE

The optimum conditions for polyphenoloxidase (PPO) extraction from endive leaves were obtained with 0.1 M phosphate buffer solution at pH 7 containing 15 mM ascorbic acid and 1% Triton X100. The major part of PPO activity, the HF2 fraction, has been purified 36 fold with a total yield of approximately 70% by ammonium sulfate precipitation and hydrophobic chromatography on Phenyl Sepharose CL4B. Two isoforms from the HF2 fraction, EC1 and EC2, have been separated by anionic exchange chromatography on DEAE-Trisacryl. Their pHi were determined by isoelectrofocusing in liquid medium and confirmed by electrophoresis on polyacrylamide gels. The pHi were 4.5 for EC2 and 5.5 for EC1. The molecular weight (Mr) determined by gel filtration was close to 120,000 for both isoforms. The optimal pH for activity was close to 6 with catechol. A small part of PPO activity, HF1, was linked to a high peroxidase (POD) activity. These two activities were not separated by concanavalin-A chromatography. The Mr determined by gel filtration was close to 46,000 and its pHi was near 8. Kinetic parameters of EC1 and EC2 appeared to be different. Among the phenolic substrates, dicaffeoyltartaric acid was the best natural substrate for endive PPO.