The transesterification of high oleic sunflower oil with butanol by the immobilized Lipozyme(R) in n-hexane was carried out in a continuous packed bed reactor, oleic acid, butyl ester, and glycerol being formed as the main products. It was found that glycerol, insoluble in n-hexane, remained in the reactor adsorbed onto the enzymatic support, leading to a drastic decrease in enzymatic activity. The phenomenon involved in this loss of activity was attributed to the formation of an hydrophilic hindrance around the enzyme resulting in diffusion limitations of the hydrophobic substrate from the organic phase to the enzyme. To recover enzymatic activity, several solutions are proposed. The addition of silica gel into the enzymatic bed to adsorb the produced glycerol did not enable this loss of activity to be avoided. In order to enhance the solubility of glycerol in the reaction medium as soon as it was produced, n-hexane amended acetone was used as solvent, but high conversion of sunflower oil was not restored. Finally, by intermittent rinsing of the catalyst bed with a solution of tertiary alcohol amended with water to obtain the optimal thermodynamic water activity of 0.54, glycerol was eliminated from the reactor, and high conversion was maintained. This semi-continuous process allowed not only the synthesis of oleic acid butyl eater but also the two products to be recovered separately.