The Proteolytic Systems of Ruminal Microorganisms Rj Wallace

The proteolytic systems of ruminal microorganisms Rj Wallace To cite this version: Rj Wallace. The proteolytic systems of ruminal microorganisms. Annales de zootechnie, INRA/EDP Sciences, 1996, 45 (Suppl1), pp.301-308. hal-00889635 HAL Id: hal-00889635 https://hal.archives-ouvertes.fr/hal-00889635 Submitted on 1 Jan 1996 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. The proteolytic systems of ruminal microorganisms RJ Wallace Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK Protein breakdown in the rumen is generally the breakdown process and assesses the regarded as detrimental to the efficiency of relative importance of different species in the ruminant nutrition, certainly for animals on a light of population densities and the properties relatively high plane of nutrition. Peptides and of the mixed rumen population. amino acids arising from proteolysis are potential nutrients for the growth of rumen microorganisms, but they are also liable to be Proteolytic ruminal microorganisms degraded to ammonia and lost from the rumen. Proteolytic activity occurs in all three main The mixed rumen microbial population has a categories of rumen microorganisms. Bacteria proteolytic activity that is only moderate are mainly responsible for dietary protein compared with other proteolytic micro- breakdown, while ciliate protozoa break down organisms and the host’s own gastric and particulate feed protein of appropriate size and pancreatic secretions, but the length of time also bacterial protein. The key features of that feed material is retained in the rumen rumen proteolytic activity are that it varies means that this activity is able to break down a greatly from animal to animal and from feed to substantial proportion of most dietary proteins feed. The predominant mechanism of peptide (0rskov and McDonald, 1979; Broderick et al, degradation is biphasic, via dipeptidyl 1991). Many strains and species of rumen aminopeptidases which cleave dipeptides from ciliate protozoa, bacteria and anaerobic fungi larger peptides followed by dipeptidase. have been found to be proteolytic, and they Dipeptidyl aminopeptidase activity occurs only contain a variety of different types of proteolytic in Prevotella ruminicola among the common enzymes (table I) (Wallace and Cotta, 1988; rumen microbial species. In contrast, Wallace, 1994). dipeptidase, which cleaves the dipeptide The predominant species of proteolytic products from dipeptidyl aminopeptidase, is bacterium found in the rumen of most animals present in many species, including P. is Prevotella (formerly Bacteroides) ruminicola, ruminicola, and is particularly high in rumen which has been identified as proteolytic in protozoa. Deamination of amino acids is many studies (Blackburn and Hobson, 1962; carried out by a combination of numerous low- Fulghum and Moore, 1963; Hazlewood and activity bacteria and protozoa and a much Nugent, 1978; Wallace and Brammall, 1985) smaller number of high-activity species. Most and which can comprise more than 60% of the ammonia production is probably carried out by flora under some circumstances (Van Gylswyk, the low-activity species, which again include P. 1990). Its cell-associated, mainly cysteine ruminicola, but proliferation of the high-activity protease activity, is fairly typical of the rumen species may be a problem on certain diets. bacterial population as a whole. Some animals The microbiology of protein breakdown in the possess Butyrivibrio fibrisolvens as the most rumen is of interest because it deals with one prevalent proteolytic isolate (Blackburn and of the major inefficiencies of ruminant nutrition, Hobson, 1962; Fulghum and Moore, 1963; namely the too-rapid conversion of protein to Hazlewood et al, 1983; Wallace and Brammall ammonia in the rumen and the subsequent 1985). B. fibrisolvens, which has a higher loss of that ammonia by absorption across the specific activity than most other species rumen wall and excretion as urea (Leng and (Wallace and Brammall, 1985), appeared to be Nolan, 1984; Wallace and Cotta, 1988; present when the dietary protein was more Wallace, 1994). This review describes the resistant to degradation (Wallace et ai, 1987). microorganisms involved at different stages of Its extracellular serine protease activity (Wallace and Brammall, 1985; Cotta and acrylamide gels (Wallace and Cotta, 1988). Hespell, 1986; Strydom et al, 1986) is not Rational manipulation of ruminal proteolysis by typical of in vivo activity. altering the proteolytic population therefore Rumen ciliate protozoa exhibit a variety of appears only a distant possibility. protease activities, the most important of which are cysteine and aspartic proteases (Forsberg et al, 1984) that have a mixture of specificities Peptidolytic rumen microorganisms with a significant trypsin-like activity (Prins et al, 1983; Forsberg et al, 1984). An Isotricha sp. Peptide breakdown to amino acids must occur had a protease profile on gel electrophoresis for the amino acids to be incorporated into that was quite different to the profile of microbial protein, and when there is sufficient Dasytricha ruminantium, and representatives of energy available to fuel biosynthesis, amino four different entodiniomorphid genera also acids will be incorporated and peptide had different protease patterns (Lockwood et breakdown would not be considered to be a al, 1988). Extracellular extracts of rumen major inefficiency in fermentation. However, ciliates were reported to have a higher activity when energy is unavailable, or when the rate of than intracellular extracts (Shinchi et al, 1986), peptide breakdown exceeds the rate at which it but although these enzymes have been can be assimilated, peptide catabolism leads to characterised biochemically (Shinchi and excessive ammonia production and poor N Kandatsu, 1983; Shinchi et al, 1986), their retention. relevance to the mixed ecosystem has not The great majority of peptidase activity in been established. rumen fluid is aminopeptidase (Wallace et al, Protozoa do not hydrolyse soluble protein 1990a). It is characterised by dipeptides rather as readily as do the bacteria. In the studies of than single amino acids being cleaved from the Ushida et al (1986, 1991 ), defaunation resulted peptide chain (table II) (Wallace et al, 1990a, in a higher activity of rumen fluid towards 1993). Enzymes of this nature are classified as soluble protein, which was believed to be dipeptidyl aminopeptidases (Webb, 1992). The caused by increased numbers of more active main mechanism of hydrolysis in intact rumen bacteria. A similar trend was seen with sheep microorganisms appeared, from the hydrolysis which had been ciliate-free from birth (Wallace of diagnostic synthetic substrates, to be et al, 1987). However, protozoa ingest protein dipeptidyl aminopeptidase type I (DAP-1 ) particles, either in the form of feed particles or (Wallace and McKain, 1989), although other bacteria, and they may be of great significance activities were also apparent. This pattern in diets containing particles of the correct differs from that obtained with sonicated dimensions. bacteria, which indicated among others a Reports of proteolytic activity associated strong X-Ala-p-nitroanilide arylamidase (Ala- with rumen anaerobic fungi are conflicting. DAP) activity (table II). The dipeptides released Whereas Neocallimastix frontalis had a high as a result of DAP activity are then broken specific activity metalloprotease (Wallace and down by separate dipeptidase activity. Ruminal Joblin, 1985), most other fungal isolates peptide breakdown is therefore a two-stage had little proteolytic activity, although process. aminopeptidase was present in all species It was established by comparing the (Michel et al, 1993). Experiments with specific activities of mixed rumen protozoa and gnotobiotic lambs indicate that fungi play a mixed bacteria prepared from rumen fluid that minor role in proteolysis in vivo (Bonnemoy et bacteria were mainly responsible for the al, 1993). breakdown of larger peptides (Newbold et al, Proteolytic activity and the microbial 1989; Wallace et al, 1990c). When pure species responsible for that activity are heavily cultures of the most common rumen bacteria diet-dependent (Nugent and Mangan, 1981; were screened for dipeptidyl aminopeptidase Siddons and Paradine, 1981; Hazlewood et al, activity, remarkably the only common bacterial 1983). However, perhaps the most remarkable species that possessed DAP-1 and had high feature of proteolysis is its inherent variability : Ala-DAP activity was P. ruminicola (table 111) different animals on the same or similar diets (Wallace and McKain, 1991). Selective and housed together had completely different isolation, using fluorogenic DAP-1 substrate patterns of proteolytic enzymes in poly- confirmed this unusual finding. P. ruminicola, like the mixed rumen population, had low Russell et al, 1988, 1991) calculated that these aminopeptidase activity against amino acyl-p- bacteria did not have sufficient activity to nitroanilide substrates but

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