Comparison of the specificity and kinetic properties of 3 milk-clotting enzymes

Abstract

SummaryThe biochemical properties of rennin purified from calf stomach,Mucor pusillusandEndothia parasiticahave been compared.The specificities of the enzymes have been studied by separating by disk-gel electrophoresis the macropeptides released on incubation with acid casein and κ-casein. The products formed on incubation with rennin from calf stomach andM. pusillusshowed some similarity although several bands were found which did not correspond to one another. The products formed with rennin fromE. parasiticadiffered considerably from those with the 2 other enzymes.The kinetic properties of the enzymes have been investigated in stopped-flow experiments on the enzyme-induced aggregation of κ-casein. The aggregation has been characterized by 2 parameters—the lag time prior to the increase in extinction and the maximum rate of change in extinction during aggregation.The specific activity measured from the inverse value of the lag time was under optimal conditions nearly 2·5 times higher for rennin from calf stomach than for rennin from the 2 micro-organisms. The fact that with increasing enzyme concentration the maximum rate of change of extinction during aggregation was identical for all 3 enzymes indicates that the same product is responsible for the formation of the aggregates. The data suggest that theKmvalue for the aggregation of κ-casein by the enzymes is about 100 times smaller thanKmfor the proteolytic activity of calfstomach rennin on κ-casein. The possibility is suggested that the high specificity of the enzymes in milk clotting is due to the very lowKmvalues for the peptide bond modifications involved in the formation of para-κ-casein, the first enzymic step in the clotting of milk.