Extraction, purification and characterisation of a milk-clotting protease from 'kesinai' (Streblus asper Lour.) leaves

Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes \nthat can serve as an alternative to calf rennet in cheese making. Different types of extraction \nbuffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH \n6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted \nusing 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, \n0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out \nusing acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. \nResults showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer \namong the buffers used in the extraction study. After the final purification step of size-exclusion \nchromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme \nshowed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was \nstable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its \noriginal activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme \nwas estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme \nwas found to be lower than that of commercial Mucor rennet.