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

Abstract

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 490C. The Vm. and the apparent Km 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 fl-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

The cellulase content of 15 species of entodiniomorphid protozoa, mixed bacteria and plant debris isolated from the ovine rumen

“…Wood et al (1982) found that 0-5 M phosphate buffer pH 7-5 gave good release of the cellulase oiRumino-coccus albus and that sonication removed 90% of that which remained. Francis, Gawthorne & Storer (1978) found that phosphate buffer released enzyme from plant solids but this was not confirmed in the present studies where gentle sonication in water, under conditions that would not normally disrupt bacteria, gave the best release of cellulase. However, there is no evidence in the present or any other studies that all the enzyme has been released from the bacteria, protozoa or plant debris, as it may still be bound in an inactive form to particulate matter.…”

The effect of dietary phosphorus and calcium level, phytase supplementation, and ileal infusion of pectin on the chemical composition and carbohydrase activity of fecal bacteria and the level of microbial metabolites in the gastrointestinal tract of pigs1,2

“…In the present study, there was no increase in the bacterial cellulase activity in feces in response to supplemental MCP as observed by Francis et al (1978) under in vitro conditions. According to these authors, rumen cellulase activity was greater when the concentration of phosphate was increased from 5 to 50 mM, and increasing Ca concentrations had no effect on cellu- lase activity.…”

Evaluation by electron microscopy and anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls

“…However, in the inocula used in the studies conducted in this laboratory, rumen fluid supernatant per se did not have marked cell wall-degrading activity without viable bacteria present as tested by TEM of tissues digested by centrifuged rumen fluid supernatant (unpublished data) and streptomycin-treated washed rumen bacteria (8). Perhaps inhibitors to the cell wall-degrading enzymes were present in the rumen fluid (29), which prevented the activity of cell-free enzymes except for those from bacteria extremely near to the forage cell walls. Although direct adherence of bacteria to certain cell walls (e.g., parenchyma bundle sheath of Bermuda grass) is required for degradation, other apparently less rigidly structured cell walls (e.g., mesophyll, parenchyma bundle sheath, and parts of the epidermis in orchard grass) appear to be available to the extracellular enzymes produced by nearby but unattached bacteria.…”

“…Results have suggested that cell wall organization, and in particular the hemicellulose component, does influence forage degradation (11,23). Furthermore, research has shown that the various carbohydrates in intact forage cell walls are not equally available to the degradative enzymes (22,29).…”