The work refers to the immobilization of the Alcaligenes sp. lipase for its use in the synthesis of biodiesel from canola oil, which has never been reported before. The enzyme was immobilized on different supports: polyethyleneimine, agarose, glutaraldehyde agarose, octyl agarose, glyoxyl agarose, Sepabeads (R) and also by aggregation and crosslinking to produced enzyme aggregates (CLEAs), so to consider different catalyst options for the reaction under study. The lipase biocatalysts were evaluated and compared based on their specific hydrolytic activities, immobilization yields and thermal stabilities under non-reactive and reactive (in the presence of oil and methanol) conditions. The lipase immobilized on Sepabeads (R) Ec-BU and CLEAs were selected to catalyze the methanolysis of canola oil. The synthesis of methyl esters from vegetable oil was performed at 40 degrees C, using oil:methanol molar ratios of 113 and 114, and also using a six-step addition of methanol to the reaction mixture (oil/methanol molar ratio of 116) in order to decrease its inhibitory effect. At oil: methanol molar ratios of 113 and 114 a maximum conversion yield of 70% was obtained after 30 h of reaction; on the other hand, using the six-step addition of methanol the maximum conversion yield obtained was of 80% in only 10 h of reaction, demonstrating that the inhibitory effect of methanol was significantly reduced. The operational stability of the catalysts was assessed in three sequential batches of 10 h each, obtaining a yield decay of 13% and 3,2% for lipase immobilized on Sepabeads (R) Ec-BU and CLEAs respectively, so the latter had a higher operational stability; however, recovery is simpler in the former so both catalyst are competitive for biodiesel production. (C) 2015 Published by Elsevier B.V.