Factors Affecting the Activity of Cellulases Isolated from the Rumen Digesta of Sheep

Sodium phosphate buffer was used to extract cellulases from the plant solids fraction of rumen contents. The mixed cellulase preparation had maximal activity at pH 6.9 and 49 degrees C. The V(max) and the apparent K(m) for wheaten hay cellulose were 19.8 glucose units/min and 6.35 mg/ml, respectively, and for microcrystalline cellulose (Sigmacell) at the same enzyme concentration, they were 33 glucose units/min and 27.5 mg/ml, respectively. For these assays a glucose unit was defined as nanomoles of glucose plus twice the nanomoles of cellobiose. Consideration of thermodynamic and kinetic data suggested that the hydrolysis of a relatively labile arabino-xylan comprising 3% of the wheaten hay cellulose was dependent on prior removal of the protecting beta-1,4-glucose chains at the outer surface of the cellulose preparation. Sequential removal of structural polysaccharides from the plant cell wall rendered the latter more susceptible to cellulase activity. Cellulase activity was stimulated by increasing the concentration of phosphate from 5 to 50 mM. The stimulation was magnified in the presence of cell-free rumen fluid. Cellulase activity was not stimulated by calcium, magnesium, iron, zinc, manganese, copper, or cobalt ions and was unaffected by the chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. O-phenanthroline inhibited activity by 30 to 50%, but this may have been due to nonchelate properties. Anaerobic conditions or thiol protective agents were not essential for either the activity or stability of the cellulases during assay. An ultrafiltrable inhibitor of cellulase activity was detected in cell-free rumen fluid.