Endothia parasitica Protease. Parameters Affecting Activity of the Rennin-like Enzyme

Abstract

Parameters expected to influence the activity of Endothia parasitica protease were investigated. Under the assay conditions using 20% nonfat dry milk dispersed in 0.2M acetate buffer, pH 5.10 and 35.0, the primary phase (initial enzymatic splitting of peptide bonds) is the rate-determining step in the clotting of milk by E. parasitica protease. Thus, the clotting time is directly proportional to the reciprocal of enzyme concentration. The higher the substrate concentration, the longer the clotting time. Between 10 and 20% substrate the relationship between the two is linear but at lower substrate concentrations the clotting time is longer than expected. The pH optimum of E. parasitica protease on acid-denatured hemoglobin and casein is 2.0 and 2.5, respectively. Over the pH range of 5.1 to 6.5 the clotting activity of E. parasitica protease is much less sensitive to the pH of the reaction medium than is that of rennin. Endothia parasitica protease has a different, and a broader, specificity than rennin and pepsin as determined on the oxidized B chain of insulin and its inability to hydrolyze the synthetic substrate, α-n-benzyloxycarbonyl-l-glutamyl-l-tyrosine.