Extraction and three-phase partitioning behavior of proteases from papaya peels

Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH4)2SO4 concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH4)2SO4 resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH4)2SO4 to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.