Isolation, Purification and Physicochemical Characterization of Polyphenoloxidases (PPO) from a Dwarf Variety of Banana (Musa cavendishii, L)

Publisher Website
Google Scholar
Abstract
A procedure for isolating and purifying polyphenoloxidase from banana pulp is described. Maximum activity was obtained from the inner portion of the pulp by extraction with 0.2M sodium phosphate buffer, pH 7.0, containing 1% insoluble PVP and 0.25% Triton X‐100. The enzyme was purified 38.8‐fold after acetone precipitation, re‐extraction of the precipitate with phosphate buffer containing Triton X‐100, storage in the freezer for 7 days (‐40°C), chromatography on Sephadex G‐100 and electrophoresis on polyacrylamide gel. The purified enzyme (four isozymes) gave a poly‐peptide molecular weight (SDS‐gel) of 31,000 ± 1,000 and molecular weight on sucrose gradient ultracentrifugation of 62,000 ± 2,000. The isoelectric point, determined by isoelectric focusing on polyacrylamide gel, was 5.2. The enzyme was stable at 55°C for 30 min; it lost mole than 90% of the activity after 5 min at 85°C and was completely inactivated at 95°C for the same time. The absorption spectrum of the purified enzyme showed a maximum at 278 nm and a shoulder at 340‐350 nm. This preparation, when submitted to paper chromatography revealed the presence of a fluorescent compound with Rf= 0.57, which was identical with chlorogenic acid.