Biodiesel production through the transesterification of oleaginous sources utilizing alkaline catalysts shows many process drawbacks like the need of a raw material with high purity degree and a high amount of alkaline wastewater generated from the biodiesel washing step. Such drawbacks can be overcome using enzymes as catalysts. In this context, the commercial Callera Trans L lipase arises as an interesting alternative catalyst for hydrolysis-esterification reaction. In order to evaluate the use of Callera Trans L on biodiesel synthesis and optimize the main variables of the hydrolysis-esterification process (temperature, methanol to oil molar ratio, and catalyst concentration), this work adopted the Central Composite Statistical Design to find out the combination of factors that lead to maximum biodiesel yield. It was observed that the Callera Trans L lipase presents a high catalytic activity, mainly in the initial reaction steps. The optimized variables for biodiesel synthesis were 35 degrees C methanol to oil molar ratio of 4.5:1, and 1.45wt% of catalyst load, which allowed reaching a biodiesel yield of 96.9 % in 24h of reaction.