Proteolytic activity of a rumen anaerobic fungus

A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn2+, Ca2+ or Co2+, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p-Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl-l-lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p-nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high-Mr form.