Polyphenoloxidase activity from strawberry fruit (Fragaria x ananassa, Duch., cv Selva): characterisation and partial purification

MARIA DE LOS ANGELES SERRADELL; PAULA ADRIANA ROZENFELD; GUSTAVO ADOLFO MARTINEZ; PEDRO MARCOS CIVELLO; ALICIA RAQUEL CHAVES; MARIA CRISTINA AÑON

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE

Society of Chemical Industry

Lugar: London, UK; Año: 2000 vol. 80 p. 1421 - 1427

0022-5142

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria x ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X-100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent Km value of 11,2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50°C and pH 5,3-6,0 without SDS and pH 7,2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7,2 but diminished it at pH 6,0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2,6-9,3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripeningstages, showing an isoelectric point of 7,3. The specific activity of PPO decreased continuosly through fruit ripening. Maximum specific activities were found at the ´´small green´ and ´large green´ ripening stages. A total enzyme extract was partially purified by means of (NH4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7,3 and a molecular mass of 135+4kDa for the native protein.