Paper Title
Three Phase Partitioning (TPP): An Easy and Cost-Effective Method for Purification of Enzymes

Currently, three-phase partitioning (TPP) has been rapidly gainingimportance with respect to separation and purification of proteins/enzymes. Various biomolecules from different sources can be purified without compromising the activity and yield. This technique involves the addition of salts ( ammonium sulphate) and alcohol (t-butanol) to the crude extract which helps in the separation of biomolecules into three distinct phasesi.e., an upper organic t-butanol phase, lower aqueous phase and middle layer having concentrated target molecules. This study focused on the purification of Polyphenol oxidase (PPO), an enzyme responsible for browning and providing resistance from biotic stress, from potatoby TPP. Phosphate buffer was used to isolate crude enzyme from mature tubers. The enzymatic assay for PPO was performed spectrophotometrically using catechol as a substrate.Prior to purification of PPO, various parameters such as ammonium sulphate saturation, extract tot-butanol ratio, temperature and pH were optimized.At 25C and pH 7.0, 40% ammonium sulphate concentration, 1:1 crude to t-butanol ratio, worked well for this enzyme.PPOwas purified up 1.64 fold with 96% recovery. PPO is a homodimer and the mol. weight of the TPP-purified PPO subunits were found to be ~20 kDa as revealed by SDS-PAGE