New insights into the molecular mechanism of methanol-induced inactivation ofThermomyces lanuginosuslipase: a molecular dynamics simulation study

Abstract

Methanol intolerance of lipase is a major limitation in lipase-catalyzed methanolysis reactions.In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solventexposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak.

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

“…It is important to note that the structural changes detected in the water-ethanol mixtures do not necessarily represent a transition toward a catalytically competent lipase. Indeed, as indicated by molecular dynamics simulations of TLL, methanol does not accommodate structural changes allowing formation of the oxyanion hole [114,115]. Hence, the changes in bimane-Trp interaction detected by TrIQ are ascribed to structural movements associated with lid-opening, but not to an active conformation.…”

Molecular Mechanism behind Solvent Concentration-Dependent Optimal Activity of Thermomyces lanuginosus Lipase in a Biocompatible Ionic Liquid: Interfacial Activation through Arginine Switch

“…Such an “activation” of the enzyme is naturally favored in a solvent possessing either a hydrophobic character or a low dielectric constant. − The separation and purification of the product become easier as well during such interfacially immobilized enzymatic catalysis (immobilized on the large hydrophobic surface of an aggregated substrate or surfactant) than that when the reaction is carried out with chemical catalysts. ,− Among all lipases, those from the thermophilic fungus Thermomyces lanuginosus (TL) are found to be industrially significant for biodiesel production, owing to their inherent stability at a high temperature, operation over a wide range of pH conditions, as well as their high methanolysis activity. − TL lipase (TLL) has been used not only in biodiesel production but also in the food industry, in the enantiomeric separation of racemic mixtures and in many regioselective or regiospecific processes to synthesize fine chemicals . Rightfully, TLL has been studied by several researchers through in vitro experiments, site-directed mutagenesis, − X-ray crystal structures, , site-directed spin labeling, intrinsic tryptophan fluorescence studies, ,− and molecular dynamics simulations. ,, …”

“…However, several reports in the literature − have also suggested that alcohol causes inactivation and/or thermal destabilization of the enzyme. A very recent simulation study of TLL in methanol has explored the details of the molecular mechanism behind this inactivation …”

“…14−17 TL lipase (TLL) has been used not only in biodiesel production but also in the food industry, in the enantiomeric separation of racemic mixtures and in many regioselective or regiospecific processes to synthesize fine chemicals. 4 Rightfully, TLL has been studied by several researchers through in vitro experiments, 18 site-directed mutagenesis, 19−21 X-ray crystal structures, 22,23 site-directed spin labeling, 24 intrinsic tryptophan fluorescence studies, 20,25−27 and molecular dynamics simulations. 19,28,29 TLL has a typical α/β hydrolase fold, 22,23 maintained by three disulfide linkages (C268−C22, C36−C41, and C104−C107).…”

“…4 Rightfully, TLL has been studied by several researchers through in vitro experiments, 18 site-directed mutagenesis, 19−21 X-ray crystal structures, 22,23 site-directed spin labeling, 24 intrinsic tryptophan fluorescence studies, 20,25−27 and molecular dynamics simulations. 19,28,29 TLL has a typical α/β hydrolase fold, 22,23 maintained by three disulfide linkages (C268−C22, C36−C41, and C104−C107). 23 Like that in all other lipases, a flexible lid (residues 86−92) covers (uncovers) the active site in the closed (open) form of the enzyme.…”