Three phase partitioning to concentrate milk clotting proteases from Wrightia tinctoria R. Br and its characterization.

Wrightia tinctoria stem proteases were partially purified for the first time through a non-chromatographic technique, three phase partitioning (TPP), to concentrate the milk clotting proteases. Various parameters like salt and solvent concentration that affect the partitioning of the protease were examined. Maximum recovery and purification fold of the protease activity were found in the interfacial phase (IP) with 60% ammonium sulphate and 1:1 crude enzyme to t-butanol. Optimum pH and temperature of the enzyme fraction were found to be 7.5 and 50 °C respectively. Inhibition studies revealed its serine nature. Non-denaturing PAGE, Zymography and 2D PAGE of IP revealed presence of three different caseinolytic proteases of molecular weights 95.62 kDa, 91.11 kDa and 83.23 kDa with pI 3.89, 5.45 and 5.43 respectively. Both aqueous and lyophilized form of IP were remarkably stable retaining complete activity at 4 °C for 3 weeks. Electrophoretic analysis of casein hydrolysate by IP at different incubation time indicated a time dependent substrate subunit specificity with hydrolysis of κ-casein commencing after 10 min followed by α and β caseins. This pattern was found similar to that by commercial vegetable coagulant, Enzeco®. Study details the effectiveness of TPP concentrated W. tinctoria proteases as a vegetable coagulant alternative in cheese making.