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Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.

Primary pathways for glucose metabolism were established in the anaerobic rumen fungus Neocallimastix frontalis EB188. This highly capable cellulolytic organism demonstrated a strict anaerobic integration of metabolic pathways. Glycolysis in N. frontalis EB188 was coupled to malate dehydrogenase, 'malic' enzyme and specified hydrogenosome reactions. Pyruvate, as in most life forms, was a pivotal compound. The major fermentation products of N. frontalis EB188 were acetate, ethanol and lactate, with the concomitant generation of H2. On the basis of its unique characteristics and streamlined fermentation pathways, it was concluded that N. frontalis EB188 should be an important contributor to programs generating energy and selected chemicals from currently intractable biomass.

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.