Milk Clotting and Blood Washing Potential of Heat-stable Acidic Serine Protease Purified from Ocimum basilicum (Sweet basil) Seeds

Abstract

 In this study for the first time we purified and characterized a serine protease of 60.5kDa purified from the mucilage and fat-free seeds of Ocimum basilicum by ammonium sulfate precipitation followed by ion exchange chromatography. Cleavage of azo-casein indicates it to be endoprotease. The enzyme exhibited a Vmax of 0.066 mM/min and a Km of 0.53 mM using BAPNA as a substrate. For enzyme activity the optimal pH was found to be 5, and the optimal temperature was found to be 40°C. The activation energy was measured at 3.82 kcal/mol, with an enthalpy change of 2.16 kcal/mol and a Q10 value of 1.20. The protease was stable within a pH range of 3 to 7 and at temperatures from 4 to 50°C. Notably, exposure to microwaves increased its proteolytic activity by 56%. Additionally, this protease showed significant effectiveness in removing blood stains when combined with sodium dodecyl sulfate (SDS) and was able to clot milk. This research represents the first characterization of a protease from Ocimum basilicum seeds, highlighting its potential applications in the detergent industry and food production, particularly in cheese making.