Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides (TAGs, DAGs, MAGs) variation during 24 h reaction. An excellent 96% FAMEs release was achieved when low (3-5%) water concentrations were used in the conversion of crude soybean oil. Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs, DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production. (C) 2013 Elsevier Ltd. All rights reserved.