Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31 kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The K-m and V-max values of the enzyme for casein were obtained to be 1.29 mg/ml and 0.035 prnol Tyr/min, respectively. Optimal temperature and pH were 65 degrees C and 5.5, respectively. After incubation of enzyme at 65 degrees C for 1 h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses. (C) 2017 Elsevier B.V. All rights reserved.