Esterification of palmitic acid and glucose by Novozyme 435 was performed in CO2-expanded acetone. The study included investigating key process parameters such as pressure, temperature, concentrations of substrate and enzyme. An amount of acetone up to 3% (v/v) is required to ensure that the reaction takes place in an expanded liquid phase. A good esterification performance could be found with 30 wt.% enzyme related to the amount of dissolved fatty acid at an optimum temperature of 50 degrees C and a pressure of 65 bar. Additionally, a new mechanism for the removal of water as a by-product of the reaction is discussed, which is due to the multi-phase distribution of acetone-CO2-water-glucose system. (C) 2008 Elsevier B.V. All rights reserved.