Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Abstract

Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastixfrontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel Rf values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases.

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188

“…With the possible exception of P-glucosidase, the intracellular activities probably represent activities similar to those in the supernatant. The temperature and pH optima of intracellular cellulases were identical or very similar to extracellular activities (5,17,18). The ion inhibitions, isoelectrofocusing values, and Rf values of cellulases were mostly similar for intracellular and extracellular samples.…”

“…Peak B4 contained ,-glucosidase exclusively. We have used these methods to purify one ,-glucosidase (Bi) (5,17,18). This 125-kDa enzyme was catalytically typical of fungal 3-glucosidases (20,25).…”

“…Proteolytic activity in the culture supernatant of N. frontalis has been reported (27) and could contribute to the diversity of cellulases found. We have provided a detailed description of protein glycosylation events in the rumen fungus N. frontalis EB188 (5,17,18). Both intracellular and extracellular proteins are highly glycosylated.…”