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

SERRADELL MA; ROZENFELD PA; MARTÍNEZ GA; PEDRO MARCOS CIVELLO; CHAVES AR; AÑÓN MC

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE

Published for Sci by John Wiley And Sons Ltd,

Lugar: Chichester; 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 powders. The presence of NaCl and Triton X-100 in the extraction buffer caused a remarkable increase of enzyme extractability. The enzyme showed an apparent Km value of 11.2 mM for pyrocatechol as substrate.The maximum enzyme activity was observed at 50°C and pH between 5.3-6.0 without SDS and 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2, while diminished the enzyme activity 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 ripening stages, showing an isoelectric points of 7.3. The specific activity of PPO continuously decreased 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 a 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±4 kDa for the native protein.   Key words: strawberry fruit; polyphenoloxidase; enzyme purification; fruit ripening.