Aqueous two-phase extraction is employed for the first time for separation and purification of mixture of enzymes (bromelain and polyphenol oxidase) from the pineapple (Ananas comosus L. Merr.). Influence of several parameters such as phase forming polyrner molecular weight (polyethylene glycol 1500-20,000), concentration of phase forming components (polyethylene glycol, 12-18%, w/w, and potassium phosphate, 14-20%, w/w) and system pH (6.0-9.0) on differential partitioning of bromelain and polyphenol oxidase required for the purification is studied. Bromelain preferentially partitioned to the top (polyethylene glycol) phase, while polyphenol oxidase to the bottom (potassium phosphate) phase. Partition coefficient of both the enzymes decreased with an increase in molecular weight of polyethylene glycol. Phase composition of the system was found to have significant effect on enzyme partitioning and degree of purification. The polyethylene glycol/potassium phosphate system (comprising of 18% PEG 1500 and 14% phosphate) resulted in about 228% activity recovery and 4.0-fold increase in purity in case of bromelain and about 90% activity recovery and 2.7-fold increase in purity of polyphenol oxidase. (c) 2007 Elsevier B.V.. All rights reserved.