Milk-clotting activity of proteinases produced by Rhizopus

Rhizopus oligosporus NRRL 3271 produces an enzyme having high milk-clotting activity. High yields of the enzyme were noted in the culture filtrates of milk, wheat flour, or wheat bran. The enzyme was stable at 40 °C, or below, but its activity was destroyed rapidly by heating at 60 °C. The enzyme was fairly stable between pH 3 and 6 and very unstable at a pH below 2 or above 7. The enzyme was recovered from the culture filtrate by ammonium sulfate fractionation (30–75% saturation). When the enzyme preparation so obtained was chromatographed on a DEAE-cellulose column and eluted by salt gradient, four active components were noted, two of which accounted for most of the activity. The NRRL 3271 enzyme and animal rennin behave similarly toward the pH of milk, added calcium chloride, and sodium chloride. Although the NRRL 3271 enzyme caused a higher degree of proteolysis than did rennin, the proteolysis apparently was not high enough to interfere with curd formation. Therefore, the NRRL 3271 enzyme merits further study as a potential replacement for rennin or as an adjunct to be incorporated into rennin for the manufacture of cheese.