Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors

Abstract

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 IS 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. Ruminal proteolysis can result in a loss of high-quality dietary protein that would otherwise be directly digested and absorbed in the small intestine of the ruminant animal (7). The first step in protein degradation in the rumen is hydrolysis of proteins by proteinases to peptides and amino acids, which are either utilized directly by the microflora or degraded further by peptidases and deaminating enzymes to shortchain fatty acids and ammonia (12, 33, 34). Proteolysis has been suggested to be the ratelimiting step in the degradation of fraction 1 protein from alfalfa (31), although with other proteins, the utilization of amino acids was considered to be the limiting step (11, 26). Methods successful in improving protein utilization in ruminants by decreasing the apparent degradation of protein in the rumen have included chemical treatment of feed materials (11), defaunation (6), and inclusion of feed additives such as monensin and diaryliodonium compounds (13, 32). The most direct and perhaps the most effective means of decreasing the degradation of protein within the rumen is through the selective

The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5

“…Our work indicated that cell-associated bovicin HC5 was inherently more resistant to degradation by Pronase E than the cell-free activity. This characteristic could be highly advantageous in a natural ecosystem like the rumen because it is inhabited by a variety of bacteria, protozoa and fungi that produce an array of proteinases (Brock et al, 1982) or peptidases (Wallace & McKain, 1991). This observation introduced yet another question.…”

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production

“…Only a few species of ruminal bacteria are actively proteolytic [15^18], but soluble proteins are degraded at a rapid rate by MRB [19]. Based on the observation that a variety proteinase inhibitors decreased the proteolytic activity of MRB, it appears that ruminal bacteria have a wide variety of proteinases [20]. Peptides arising from proteolysis can be utilized by ruminal bacteria [21,22], but peptides are also degraded extracellularly by peptidases [23].…”

Yield improvement for manufacture of α1‐proteinase inhibitor

“…Alpha1-PI activity was measured as described previously [5]. Azocaseinolysis was assayed at 5 ° C and pH 8·1 or 9·3 by standard procedure [6]; linear kinetics were obtained during the time course of the assay. Amidolysis was assayed at 0 ° C and at pH 8·1 or 9·3 using S-2288 (DiaPharma Group, Inc., Franklin, OH) as substrate [7]; linear kinetics were obtained during the time course of the assay.…”