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Polyphenoloxidase activity from strawberry fruit ( Fragaria ananassa , Duch., cv Selva): characterisation and partial purification
Author(s) -
Serradell María de los Angeles,
Rozenfeld Paula Adriana,
Martínez Gustavo Adolfo,
Civello Pedro Marcos,
Chaves Alicia Raquel,
Añón María Cristina
Publication year - 2000
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/1097-0010(200007)80:9<1421::aid-jsfa649>3.0.co;2-k
Subject(s) - isoelectric point , chemistry , ripening , fragaria , isoelectric focusing , chromatography , enzyme assay , enzyme , acetone , fast protein liquid chromatography , extraction (chemistry) , substrate (aquarium) , food science , high performance liquid chromatography , botany , biochemistry , biology , ecology
In this work, polyphenoloxidase (PPO) from Selva strawberry fruit ( Fragaria × 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 K m value of 11.2 m M 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 specific activity of PPO decreased continuously 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 (NH 4 ) 2 SO 4 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 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry