Three Phase Partitioning (TPP) is reported here for the first time for beta-galactosidase purification in single step and two step processes. The most favourable ammonium sulphate concentration (30% w/v), crude to t-butanol ratio (1:0.5) and temperature (25 degrees C) resulted in 78% recovery with 7.5-fold purity of beta-galactosidase in the single step process. Enhanced fold purity (21 times) with an enzyme recovery of 72% was achieved at the aforementioned temperature (25 degrees C) and crude to t-butanol ratio (1:0.5) with 15-60% ammonium sulphate saturation in the second step. Characterisation showed that this purified enzyme exhibited a double pH optimum (3.0 and 7.0) that was more pronounced when lactose was used as the substrate. The enzyme was further characterised with respect to its optimum temperature, energy of enzyme activation and deactivation, kinetic constants (V-max, and K-m) and pK(1) and pK(2) values and effect of monovalent and divalent cations. (C) 2013 Elsevier B.V. All rights reserved.