• Energy Research

Lipase‐catalyzed transesterification of triglycerides and alcohols to obtain biodiesel is an environmentally friendly and sustainable route for fuels production since, besides proceeding in mild reaction conditions, it allows for the use of low‐cost feedstocks that contain water and free fatty acids, for example non‐edible oils and waste oils. This review article reports recent advances in the field and focus in particular on a major issue in the enzymatic process, the inactivation of most lipases caused by methanol, the preferred acyl acceptor used for alcoholysis. The recent results about immobilization of enzymes on nano‐materials and the use of whole‐cell biocatalysts, as well as the use of cell‐surface display technologies and metabolic engineering strategies for microbial production of biodiesel are described. It is discussed also insight into the effects of methanol on lipases obtained by modeling approaches and report on studies aimed at mining novel alcohol stable enzymes or at improving robustness in existing ones by protein engineering.

Lipases are widely used enzymes that catalyze hydrolysis and alcoholysis of fatty acid esters. At high concentrations of small alcohols such as methanol or ethanol, many lipases are inhibited by the substrate. The molecular basis of the inhibition of Candida antarctica lipase B (CALB) by methanol was investigated by unbiased molecular dynamics (MD) simulations, and the substrate binding kinetics was analyzed by Markov state models (MSMs). The modeled fluxes of productive methanol binding at concentrations between 50 mM and 5.5 M were in good agreement with the experimental activity profile of CALB, with a peak at 300 mM. The kinetic and structural analysis uncovered the molecular basis of CALB inhibition. Beyond 300 mM, the kinetic bottleneck results from crowding of methanol in the substrate access channel, which is caused by the gradual formation of methanol patches close to Leu140 (helix α5), Leu278, and Ile285 (helix α10) at a distance of 4-5 Å from the active site. Our findings demonstrate the usefulness of unbiased MD simulations to study enzyme-substrate interactions at realistic substrate concentrations and the feasibility of scale-bridging by an MSM analysis to derive kinetic information.