Optimization of three-phase partitioning system for the purification of protease from Calotropis gigantea latex using response surface methodology

The response surface methodology (RSM) was utilized to optimize three-phase partitioning (TPP) parameters for the purification of Calotropis gigantea latex protease. The limits of purification parameters namely saturation of ammonium sulfate salt, volume ratio of tert-butanol to crude latex extract and pH value were selected from single-factor experiments. A Box-Behnken design was employed to determine the effects of TPP parameters on protease purification. From the optimization study, the degree of purification and recovery percentage reached maximum values of 6.7 and 126.12%, respectively, under optimized parameters of 52% saturation of (NH4)(2)SO4 (w/v), 1.0:1.5 ratio of crude latex extract to tert-butanol (v/v) and pH 6. The concentrated protease in the interfacial phase (IP) demonstrated higher enzyme activity when compared to the lower aqueous phase (AP). The molecular weight of recovered protease was 24 kDa from electrophoretic analysis, and the substrate zymography revealed the presence of protease in IP fraction. The findings of this study demonstrated that TPP is an effective and economic protocol for quick recovery of latex protease in comparison with expensive and time-consuming chromatographic techniques.