A silica gel-based substrate feeding system was developed to prevent methanol inhibiting the catalyst during enzymatic biodiesel synthesis. In the system, silica gel swelled upon methanol addition and subsequently released it in a controlled manner to prevent excess methanol affecting the enzyme. Biodiesel was synthesized by the enzymatic transesterification of canola oil with methanol. For this reaction, enzyme loading, methanol/oil molar ratio, silica gel dosage, glycerol content, and methanol feeding method were tested using commercial immobilized enzymes (Novozym 435 and Lipozyme RM IM from Novozymes). The results showed that conversion was highest with controlled substrate feeding rather than direct methanol addition, suggesting that the method developed here can easily prevent enzyme inhibition by limiting methanol concentration to an acceptable level. (C) 2011 Elsevier Inc. All rights reserved.