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

SERRADELL, M.A; ROZENFELD, P.A; MARTINEZ, G.A; CIVELLO, P.M; CHAVES, A.R; AÑÓN, M.C

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE

JOHN WILEY & SONS LTD

Año: 2000 vol. 80 p. 1421 - 1427

0022-5142

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragariaananassa, Duch) was extracted, characterised and partially puri®ed. 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 11.2mM 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 ripening stages, showing an isoelectric point of 7.3. The speci®c activity of PPO decreased continuously through fruit ripening. Maximum speci®c activities were found at the `small green´ and `large green´ ripening stages. A total enzyme extract was partially puri®ed by means of (NH4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The puri®cation grade achieved was near 25. The partially puri®ed enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 1354kDa for the native protein.Fragariaananassa, Duch) was extracted, characterised and partially puri®ed. 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 11.2mM 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 ripening stages, showing an isoelectric point of 7.3. The speci®c activity of PPO decreased continuously through fruit ripening. Maximum speci®c activities were found at the `small green´ and `large green´ ripening stages. A total enzyme extract was partially puri®ed by means of (NH4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The puri®cation grade achieved was near 25. The partially puri®ed enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 1354kDa for the native protein.Km value of 11.2mM 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 ripening stages, showing an isoelectric point of 7.3. The speci®c activity of PPO decreased continuously through fruit ripening. Maximum speci®c activities were found at the `small green´ and `large green´ ripening stages. A total enzyme extract was partially puri®ed by means of (NH4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The puri®cation grade achieved was near 25. The partially puri®ed enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 1354kDa for the native protein.°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 ripening stages, showing an isoelectric point of 7.3. The speci®c activity of PPO decreased continuously through fruit ripening. Maximum speci®c activities were found at the `small green´ and `large green´ ripening stages. A total enzyme extract was partially puri®ed by means of (NH4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The puri®cation grade achieved was near 25. The partially puri®ed enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 1354kDa for the native protein.4)2SO4 precipitation and cationic exchange chromatography in an FPLC system. The puri®cation grade achieved was near 25. The partially puri®ed enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 1354kDa for the native protein.4kDa for the native protein.