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Preparation and Some Basic Properties of Cell-Free Cellulolytic Extracts of Rumen Fluid^{1, 2,}

Department of Dairy Science, The Pennsylvania State University, University Park

ABSTRACT

A method was developed by which cell-free cellulolytically active extracts were obtained from rumen fluid. The procedure consisted of agitating rumen fluid in a Waring blendor, followed by high-speed centrifugation to remove bacterial cells and feed particles. Rumen fluid of pH 6.0 yielded the strongest cellulolytically active extracts. Extracts obtained by this method were active on carboxymethylcellulose (CMC 70-L, a soluble cellulose) and exhibited some activity on Whatman cellulose powder (an insoluble cellulose). The optimum pH for the activity of the extracts was 5.5–6.0. Erlenmeyer flasks as incubation vessels facilitated greater cellulose hydrolysis than did test tubes. Two grams of CMC as substrate appeared to be optimum when incubated in 250-ml. flasks containing 10 ml. of extract and 10 ml. of phosphate-citrate buffer. Rates of reaction trials showed maximum rates of hydrolysis immediately after adding the substrate, with little activity after the 16th hr. of incubation. Fermenting rumen fluid with cellulose substrate before making the extraction decreased the activity of the extract. Reducing sugar accumulated in flasks to which no bacterial inhibitor had been added, provided sterile conditions were maintained. The addition of either glucose or glucose and cellobiose did not decrease the rate of CMC hydrolysis. However, paper chromatograms showed that a sugar corresponding to cellobiose was produced in the flasks to which only glucose had been added.

¹ Authorized for publication on August 18, 1958, as Paper No. 2286 in the Journal Series of the Pennsylvania Agricultural Experiment Station.

² Taken from data presented by the senior author to the Graduate School of the Pennsylvania State University, in partial fulfillment of the requirements for a M.S. degree.