Investigating the structural properties of the active conformation BTL2 of a lipase from Geobacillus thermocatenulatus in toluene using molecular dynamic simulations and engineering BTL2 via in-silico mutation

Abstract

The discovery or development of thermoalkalophilic lipases that show high levels of catalytic activity in organic solvents would have important industrial ramifications. However, this goal is yet to be achieved because organic solvents induce structural changes in lipases that suppress their catalytic abilities. A deep understanding of these structural changes to lipases in the presence of organic solvents is required before strategies can be devised to stop them from occurring. In this work, we investigated the effects of an organic reaction medium, toluene, on the structure of the Bacillus thermocatenulatus lipase BTL2 using MD simulation. The main aims were to identify the regions of the protein that are particularly sensitive to the presence of an organic solvent, and how the presence of a hydrophobic medium affects the overall stability of the enzyme. Upon analyzing how the behavior of the enzyme differed in aqueous and hydrophobic media, it was found that many significant zones of the protein suffer in the presence of an organic solvent, which increases the rigidity of the system. This was readily apparent when we investigated important noncovalent interactions (salt bridges) and probed how distances between the atoms of the catalytic triad Ser114, Asp318, and His359 change in the presence of toluene. Moreover, the high tendency for the system to destabilize in toluene was explained by the results of FoldX calculations. Calculations showed that the addition of a small amount of water to the hydrophobic reaction environment should restore the required flexibility of BTL2. The insights gained from the analysis of our simulations allowed us to propose a modification of BTL2, the G116P mutation, that should result in the structural behavior of BTL2 in organic solvent being closer to that of BTL2 in water.

Performance Comparison of Commercial and Home-Made Lipases for Synthesis of Poly(δ-valerolactone) Homopolymers

“…The decreases in monomer conversions may be resulted from inefficient enzyme-solvent interactions or water incorporated from atmosphere. It is known that, lipases catalyze esterification and transesterification reactions in hydrophobic organic solvents, whereas they catalyze hydrolysis in aqueous media [19]. Additionally, polydispersity indexes were measured around 1.5 for both two immobilized CALB preparations by GPC analysis.…”

Biocatalysis at Extreme Temperatures: Enantioselective Synthesis of both Enantiomers of Mandelic Acid by Transesterification Catalyzed by a Thermophilic Lipase in Ionic Liquids at 120 °C

“…Particularly, the use of an apolar organic solvent (toluene) has been reported to increase BTL2 rigidity, as deduced from molecular dynamics simulations at temperatures as high as 450 • K (176.85 • C) [72], without observing any tendency for the lid to open. These same authors described that either inserting a thin layer of water around the enzyme or promoting a single point mutation (G116P) would be required for retaining activity in acyl-transfer processes [72].…”

“…Particularly, the use of an apolar organic solvent (toluene) has been reported to increase BTL2 rigidity, as deduced from molecular dynamics simulations at temperatures as high as 450 • K (176.85 • C) [72], without observing any tendency for the lid to open. These same authors described that either inserting a thin layer of water around the enzyme or promoting a single point mutation (G116P) would be required for retaining activity in acyl-transfer processes [72]. Additionally, another very recent study confirmed the reduced flexibility of BTL2 in nonpolar organic solvents, using molecular dynamics on toluene and cyclohexane, and thus confirming the enhancement of thermostability of BTL2 in the presence of these type of solvents [38].…”

“…Additionally, chemical [55,58,[65][66][67] and genetic [56,[68][69][70][71] modifications of BTL2 lipase for improving its catalytic behavior (typically, to reduce the steric hindrance around the active site) have been also reported. Finally, different papers in recent literature have employed the reported 3D structure of BTL2 for performing molecular simulations aiming to rationalize its catalytic performance and stability [38,[72][73][74].…”

Biocatalysis at Extreme Temperatures: Enantioselective Synthesis of both Enantiomers of Mandelic Acid by Transterification Catalyzed by a Thermophilic Lipase in Ionic Liquids at 120 °C

“…In fact, for BTL2 it has been reported, based on molecular dynamics simulation, how the use of an apolar organic solvent (toluene) made the lipase structure more rigid, even in simulations carried out at 450°K (176.85°C) [90], without observing any tendency for the lid to open, and claiming that either inserting a thin layer of water around the enzyme or promoting a single point mutation (G116P) would be required for retaining activity in acyl-transfer processes [90]. Additional, another very recent study confirmed the reduced flexibility of BTL2 in non-polar organic solvents, using molecular dynamics on toluene and cyclohexane, and thus confirming the enhancement of thermostability of BTL2 in the presence of this type of solvents [53].…”