Characterization of Extracellular Proteolytic Activity From Prevotella Ruminicola and Streptococcus Bovis

Abstract

The hypothesis of the study was that by isolating and characterizing proteinases from two of the predominant bacterial species in the rumen, Prevotella ruminicola and Streptococcus bovis, the understanding of the breakdown of dietary N in the rumen would be improved. Studies of how Prevotella ruminicola B$\sb1$4 degraded whole protein and utilized the hydrolytic products for growth suggested that P. ruminicola might be more important in ruminal peptide breakdown rather than protein breakdown. Further examination of the proteolytic activities of P. ruminicola NCFB 2202, D31d, GA33, M384, and B$\sb1$4 revealed a range of proteolytic enzymes varying in number, size, and catalytic type. Large differences in total proteolytic activity were observed when these strains were grown with media containing clarified rumen fluid. Subsequent investigations with Streptococcus bovis KEG, JB1, NCFB 2476, and K11.21.09.6C showed a high degree of similarity in the proteolytic activities with little variation in number, size and catalytic type. The proteolytic activity of S. bovis KEG was partially purified to reveal serine proteases of high molecular weight (160-190 kDa). Oligonucleotide primers were developed to locate and clone the serine protease gene of S. bovis KEG. The differences in the level and type of proteolytic activity exhibited by P. ruminicola strains grown under similar conditions makes the generalization that the P. ruminicola species is a prominent ruminal proteolytic bacterium questionable, because current research can not determine the proportion of the ruminal bacterial population occupied by individual P. ruminicola strains. In contrast, the similarity of the proteolytic activities of individual S. bovis strains would support the generalization that the S. bovis species is a prominent ruminal proteolytic bacterium, because the overall proteolytic activity of the S. bovis population would be less affected by changes in the proportions of individual strains. Further research is warranted to improve the understanding of molecular biology and genetics of ruminal proteolysis.