Enzymatically active cell wall isolated from olive (Olea europaea) fruit was employed to investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases, beta-glucosidase (EC 3.2.1.21), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23), alpha-arabinosidase (EC 3.2.1.55), alpha-mannosidase (EC 3.2.1.24), beta-xylosidase (EC 3.2.1.37) and beta-N-acetylglucosaminidase (EC 3.2.1.30), as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15), were identified in the cell wall preparation, at four stages of ripeness (mature green, changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes (ionically and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase), and were compared to those of forms solubilized from acetone powders with 500 mM citrate buffer (cytoplasmic and/or apoplastic plus ionically bound to cell wall). In general, the activities of low ionic strength buffer soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-bound glycosidase. During ripening there was a marked change in the percentages of covalently- and ionically linked activities of beta-glucosidase and beta-galactosidase; at the changing colour stages about 75-80% of the bound activity was present in high ionic strength buffer while at the black-ripe stage it was only 15-20%. A possible role for these cell wall degradative enzymes in olive softening is discussed.
