Major drawbacks for enzyme-catalyzed transesterification of vegetable oil triglycerides to produce biodiesel (esters of short chain alcohols) are the high concentration of alcohol in the reaction mixture and the deposition of the produced glycerol on the enzyme support. Although stepwise addition of alcohol to the reaction mixture solves the first problem, an effective way to remove glycerol from the reaction system is needed. In the present study the efficiency of three systems for removal of glycerol from the reaction mixture during the enzymatic synthesis of sunflower oil methyl esters was investigated. By coupling a single cold column to the reactor 87.6% of total glycerol produced by the reaction was removed, while 10.9% was still retained by the enzyme. The percentage of glycerol separated from the reaction system increased to 93.1% when a second separation column packed with Amberlite BD10DRY resin was included, reducing the percentage of glycerol retained by the enzyme to 5.8%. Finally, changing the geometry of cold separation column in which a conical decanter was used, the percentage of glycerol separated increased to 99.2%, while only 0.4% was retained by the enzyme. Results demonstrated that use of a convenient separation system such as those described before, glycerol produced during transesterification of sunflower oil can be efficiently removed from the reaction mixture. In this way the well-documented inhibitory effects of the glycerol product are controlled and enzymatic catalysis remains fully functional. (C) 2015 Elsevier Ltd. All rights reserved.