Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors

Proteolytic activity of the bovine rumen microflora was studied with azocasein as the substrate. Approximately 25% of the proteolytic activity of rumen contents was recovered in the strained rumen fluid fraction, and the balance of the activity was associated with the particulate fraction. The proportion of proteinase activity associated with particulate material decreased when the quantity of particulate material in rumen contents was reduced. The specific activity of the proteinase from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction. Proteinase inhibitors of synthetic, plant, and microbial origin were tested on proteolytic activity of the separated bacteria. Synthetic proteinase inhibitors that caused significant inhibition of proteolysis included phenylmethylsulfonyl fluoride, N-tosyl-1-lysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone, EDTA, cysteine, dithiothreitol, iodoacetate, and Merthiolate. Plant proteinase inhibitors that had an inhibitory effect included soybean trypsin inhibitors types I-S and II-S and the lima bean trypsin inhibitor. Proteinase inhibitors of microbial origin that showed an inhibitory effect included antipain, leupeptin, and chymostatin; phosphoramidon and pepstatin had little effect. We tentatively concluded that rumen bacteria possess, primarily, serine, cysteine, and metalloproteinases.