Action of rennet and other proteolytic enzymes on casein in casein-agar gels

Summary A series of precipitation zones appears when proteolytic enzymes diffuse through an agar gel containing 1% whole casein and 10–20 mM-calcium chloride. As the enzyme diffuses through the gel the casein stability changes giving first a precipitate which subsequently redissolves followed by a second precipitate which also redissolves. The first or outermost zone of precipitation occurs at the periphery of the area of the diffused enzyme. Using a high enzyme concentration all four zones, two of precipitation and two of subsequent clearing, are visible in about 24 h. The proportion of the total nitrogen which occurs as non-protein nitrogen (N.P.N.) in the first or outermost precipitation zone is similar to that found after the first stage of rennet action on casein solutions.