Three-phase partitioning (TPP) was used to purify invertase from Baker's yeast (Saccharomyces cerevisiae) in a single step. The influence of various process parameters like ammonium sulfate concentration, crude extract to t-butanol ratio and pH on invertase partitioning is investigated in order to enhance the purification fold degree and activity recovery of invertase. Under optimized conditions (50%, w/v, ammonium sulfate saturation, 1:0.5 crude extract to t-butanol ratio and pH 4.0), the invertase was purified to 15-fold with 363% activity recovery. The enzyme obtained from TPP showed considerable purification on sodium dodecyl polyacrylamide gel electrophoresis with a molecular weight of 52 kDa. The optimum temperature and pH were determined as 60 degrees C and pH 6.0, respectively. The purified enzyme was also very stable at a pH range of 4.0-6.5 and temperature range of 4-50 degrees C. The K-m and V-max values were calculated from Lineweaver-Burk plot as 0.19 M and 29.8 U, respectively. (C) 2010 Elsevier B.V. All rights reserved.