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

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 extr action buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K-m value of 11.2 mM with pyrocatechol as substrate. Th e maximum enzyme activity was observed at 50 degrees C and pH 5.3-6.0 witho ut 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 the rmal stability and maintained activities equal to or greater than 50% of it s maximum activity in the 2.6-9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelect ric point of 7.3. The specific activity of PPO decreased continuously throu gh fruit ripening. Maximum specific activities were found at the 'small gre en' and 'large green' ripening stages. A total enzyme extract was partially purified by means of (NH4)(2)SO4 precipitation and cationic exchange chrom atography in an FPLC system. The purification grade achieved was near 25. T he partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135+/-4kDa for the native protein. (C) 2000 Society of C hemical Industry.