CHARACTERIZATION OF CHITINASES AND BETA-1,3-GLUCANASES IN GRAPEFRUIT FLAVEDO DURING FRUIT-DEVELOPMENT

Chitinase (EC 3.2.1.14) and beta-1,3-glucanase (EC 3.2.1.39) activitie s in the flavedo of grapefruit (Citrus paradisi cv. Marsh) were determ ined at 17 times during the course of fruit development. Chitinase act ivity is initially high in flavedo, but drops rapidly and is low, alth ough fairly constant throughout the remainder of fruit development. In contrast to chitinase, beta-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flaved o extracts following SDS-PAGE. were probed with antibodies raised agai nst purified citrus chitinase and beta-1,3-glucanase. Results of immun ostaining revealed that changes in the activities of chitinase and bet a-1,3-glucanase were reflected in the amount of chitinase and glucanas e protein present in the extracts. Only a single chitinase band was de tected on western blots of crude flavedo extracts, whereas one glucana se 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 enzym es were present in the extracts; acidic and basic forms of chitinase w ere present in nearly equal amounts whereas basic glucanases predomina ted (91% of total activity). Acidic and basic chitinases differed in s ubstrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of beta-1,3 linkages for activity, were active against both soluble and insoluble beta-1,3 glucans and generated simi lar products.