Characterization of chitinases and β‐1,3‐glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit (Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.