title: Characterization of proteases formed by Bacteroides fragilis.
abstract: Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease:arylamidase ratios of these enzymes indicated that they were probably endopeptidases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 degrees C. Proteolysis was temperature sensitive, however (90% reduction at 60 degrees C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaCl suggested that ionic interactions were involved in the association of these enzymes with the membranes.
authors.affiliation: NRC Dunn Clinical Nutrition Centre, Cambridge, UK.
authors.forename: S A
authors.initials: SA
authors.lastname: Gibson
authors.forename: G T
authors.initials: GT
authors.lastname: Macfarlane
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors; D001441:Bacteroides fragilis / Q000201:enzymology*; D002412:Cations / Q000378:metabolism; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000378:metabolism; D017830:Octoxynol; D010447:Peptide Hydrolases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013696:Temperature
chemical_list: D002412:Cations; D011092:Polyethylene Glycols; D017830:Octoxynol; D009113:Muramidase; D010447:Peptide Hydrolases; D000626:Aminopeptidases