Crude canola oil (CCO) contains about 100-300 ppm of phospholipids, which have shown negative effects on biodiesel/buffer solution phase separation, resulting in low biodiesel production yield. Therefore, phospholipids should be removed before transesterification by a degumming process for efficient production of biodiesel. In this study, two-step enzymatic processes (degumming and transesterification) were carried out for the production of biodiesel from CCO. Degumming of CCO was performed using phospholipase A2 as a degumming reagent. The initial phospholipid content was reduced to less than 5 ppm by enzymatic degumming. The effects of three formulations of enzyme catalyst on the efficiency of transesterification were investigated. As a result, conversion rates of degummed CCO to fatty acid methyl esters (FAME) were 68.56%, 70.15%, and 84.25%, respectively. Lipase formulation composed of a 1:1 (vol:vol) enzyme mixture of Rhizopus oryzae and Candida rugosa showed the best performance among those tested. In order to recover and reuse the lipase catalyst efficiently, a 1:1 enzyme mixture of R. oryzae and C. rugosa was immobilized on silica gel. The immobilized lipase was used in subsequent transesterification optimization experiments. Optimization of transesterification was performed by response surface methodology (RSM). A total of 20 experiments based on RSM were carried out, and the optimal reaction conditions appeared to be 24.4% (w/w) immobilized catalyst, 13.5% (w/w) buffer solution, and 15.8% (w/w) methanol based on oil mass. Conversion rate of degummed CCO to FAME was determined to be 88.9% under optimal conditions. (C) 2011 Elsevier Ltd. All rights reserved.