The effect of the composition of the feedstock (free fatty acids and methanol), the presence or absence of moisture and the contact with biodiesel on the catalytic, enzymatic and physical stability of Novozym (R) 435 during enzymatic biodiesel production was studied. The continuous removal of moisture leads to the deactivation of the biocatalyst and favours the degradation of the polymethylmethacrylate (PMMA) that constitutes the support of the lipase B of Candida antarctica (CALB). Nevertheless, the presence of PMMA along with the active protein was detected in the reaction media regardless of the operating conditions. Surprisingly, biodiesel (without the presence of methanol) is able to diffuse inside the biocatalyst's beads producing its swelling, modifying the internal texture and also dissolving the polymeric matrix. (C) 2013 Elsevier B. V. All rights reserved.