CONICET | Buscador de Institutos y Recursos Humanos

Worldwide increasing cheese demand has led to the search of novel proteases with milk clotting activity. Plants are used as source of proteases due to their easy availability, efficient purification processes and isolation of natural coagulant. StAP1 and StAP3 are two aspartic proteases (APs) from potato tubers and leaves with milk-clotting and caseinolytic activity. The aim of this work was to optimize the caseinolytic activity of StAP1 y StAP3 and to test the specific activity on bovine casein subunits. Box-Behnken design with three factors at three different levels was used to optimize the caseinolytic activity. Second order models were used to generate response surface contours (RSC). Optimum condition determined for caseinolytic activity were: pH 8,20, temperature 43,19ºC and concentration 5,20x10-7 M for StAP1, and pH 8,21, temperature 42,68ºC and concentration 5,16x10-7 M for StAP3. These conditions are compatible with ones required for industrial cheese making process.Additionally, we evaluated the specific activity of StAP1 and StAP3 on casein subunits (α, β and κ). In order to do that, we incubate each StAP with casein subunits for 2 h and the degradation patterns were analyzed by SDS-PAGE. Densitometric analysis were performed using Scion Image. Results obtained shown that β-casein was degraded by both APs in a major proportion than others casein subunits (34% for StAP1 and 66% for StAP3). The percentages of degradation to α-casein were 25% and 36%, to StAP1 and StAP3, respectively. Finally, κ-casein was degraded 18% for StAP1 and 23% for StAP3. These results suggest that StAP1 y StAP3 degrade mainly β-casein. However, the minor degradation of κ-casein is enough to inducing milk clotting. Based on this results StAP1 y StAP3 might be considered a promising alternative of natural calf rennet for the coagulation of milk leading to new dairy products.Keywords: proteolytic enzymes, milk-clotting activity, plant proteases, cheese making.