Caseinolytic and milk-clotting activities of a new protease from Ficus johannis and its enzymatic activity on the different substrate

Coagulation of milk is the main step for producing cheese, and coagulating enzymes have been utilized for centuries in cheese making. Chymosin (EC 3.4.23.4), as the main enzymatic compound in calf rennet, has been widely used as a coagulating agent in cheese making. Regarding the high price of calf rennet as well as ethical considerations of its, a great deal of effort has been made to find appropriate milk coagulant substitutes produced either in plants or microorganisms. Here, we evaluated the caseinolytic and milk-clotting activitiesof a protease from Ficus johannis. The protease with milk-clotting activity was purified and the caseinolytic and milk-clotting activities were assessed using casein and skim milk as a substrate, respectively. The effects of protease inhibitors on milk-clotting activity were also reported. Additionally, the enzymatic activity of the purified protease on the other different substrates was examined. The protease was purified 9-fold with 44% activity recovery. The enzyme was strongly inhibited by iodoacetamide, a typical cysteine protease inhibitor, indicating that it belongs to the cysteine protease family. The highest caseinolytic activity was detected at pH 6.5 and 60°C temperature. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and β-casein. According to the present study, a novel milk-clotting cysteine protease from F. johannis was characterized. The high degree of hydrolysis on casein, in particular on κ-casein, high milkclotting activity and high clotting activity/proteolytic activity ratio of the isolated enzyme could be useful in the dairy industry.