Three phase purification of milk clotting protease from Wrightia tinctoria fruit and studies on its casein subunit specificity

Crude aqueous extract of Wrightia tinctoria fruit was subjected to three phase partitioning (TPP) with three different ammonium sulphate concentrations (40, 60, 80%) and their effect on concentrating milk clotting proteases were examined with 1:1 ratio of crude extract to t-Butanol. Proteases that concentrated in the interphase (IP) fraction of 60 and 80% salt concentration exhibited high milk clotting activity. TPP contributed to reduction in Caseinolytic Activity (CA) and enhancement of Milk Clotting Index (MCI) in these IP fractions compared to the crude enzyme. Casein (whole and kappa) hydrolytic pattern was analyzed by tricine SDS PAGE. Electrophoretic pattern of hydrolysate after 1 hr incubation revealed hydrolysis of κ-CN by CE, IP 60 and IP 80 fractions. Appearance of two low molecular weight bands approximately around 15 kDa indicated the specific affinity and controlled hydrolytic behavior by the proteases. Study provides evidence towards the utility of TPP in concentrating these milk clotting proteases and in improving their MCI.