Three-phase partitioning of protease from Calotropis procera latex

Three-phase partitioning (TPP) was used to partially purify protease from the latex of Calotropis procera (C. procera). To optimize the TPP for protease isolation a ratio of crude extract to t-butanol, percent saturation of (NH4)(2)SO4, and the cycle of TPP was required. The highest proteolytic recovery (first cycle) of 182% with a purification of 0.95 folds was obtained at the interphase of the system comprising the ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 50% (NH4)(2)SO4. The second cycle of TPP was prepared by adding of (NH4)(2)SO4 up to 65% (w/v) to the bottom phase obtained from 30% (NH4)(2)SO4-1.0:0.5 system of the first TPP. A purification of 6.92-fold was achieved with about 132% activity recovery. SDS-PAGE and zymography profiles revealed the substantial isolation of protease from C. procera latex by the TPP. The molecular weight of major protease was found to be around 28 kDa. The present study shows high interesting outcomes and could be used as a primary purification process in comparison with existing literature's values. (C) 2010 Published by Elsevier B.V.