PEPTIDASES OF THE RUMEN BACTERIUM, PREVOTELLA-RUMINICOLA

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacte rium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic amin opeptidase substrates by sonicated extracts and whole cells of differe nt species of rumen bacteria indicated that P. ruminicola had the grea test range and specific activity of dipeptidyl peptidases among the sp ecies tested. Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala(2)- p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptide s from the amino-terminus of longer peptides, were much more active th an aminopeptidases removing single amino acids in P. ruminicola. Ion-e xchange chromatography of sonicated extracts of P. ruminicola M384 rev ealed at least four distinct activities: one hydrolysed Ala(2)-p-nitro analide, ValAla-p-nitroanilide, Ala(4) and Ala(5); another was an O-2- sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4- methoxynaphthylamide, Gly(5) and ValGlySerGlu, similar to dipeptidyl p eptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and G lyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase ty pe IV DPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All o f the enzymes, and particularly those active against Ala(2)-p-nitroani lide and GlyPro-p-nitroanilide, were inhibited by serine protease inhi bitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and th e enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited stro ngly by iodoacetate. DPP-Q was inhibited completely by diprotin A. Com petitive inhibition experiments suggested that DPP-1 was less importan t than the other enzymes in the breakdown of peptide mixtures. (C) 199 7 Academic Press.