The performance of lipase NS 40116, a novel and promising soluble enzyme obtained from modified Thermomyces lanuginosus microorganism, was investigated in the production of biodiesel (fatty acid methyl esters-FAME) by hydroesterification. In order to investigate the potential of the biocatalyst in its soluble form, this work reports the effect of water content and enzyme load, as well as the recovery and reuse of the biocatalyst. A FAME yield of 94.30% after 12h was achieved at 35 degrees C by combining 0.50wt% of lipase, 15wt% of water, and a methanol:oil molar ratio of 4.5:1. The analysis of the time course reaction suggests that the triacylglycerides (TAGs) are hydrolyzed by the enzyme in a first step, generating free fatty acids (FFAs), followed by the esterification of these FFAs into FAME. In relation to the reusability assays, the lipase kept approximately 90% of its catalytic activity after five cycles of reuse. In this context, the findings of this study demonstrate that lipase NS 40116 can efficiently catalyze hydroesterification reactions under mild conditions, arising as a competitive alternative for biodiesel synthesis.