Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Abstract

Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a(w)) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein-bound water molecules increased linearly with a(w), until at a(w)=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate-binding site. At higher water activity, the thickness of the hydration shell increased up to 10 A close to a(w)=1. Above a limit of 1600 protein-bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate-binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.

Solvent-Free Lipase-Catalyzed Synthesis of Technical-Grade Sugar Esters and Evaluation of Their Physicochemical and Bioactive Properties

“…The saccharide concentration in the liquid phase of the reaction medium decreased during the time course of reaction due to its consumption by the reaction; however, saccharide was not completely consumed ( Figure 1C). It appears that the ultra-low water concentration did not negatively affect CALB activity, in agreement with the results in literature [20,24]. As shown in Figure 1D, the proportion of monoester decreased during the time course of both reactions, from 90% to 60%-70%.…”

Lipase hydration state in the gas phase: Sorption isotherm measurements and inverse gas chromatography

“…This assumption is highlighted at a w = 0.2, where the curve displays a break point whereas a linear curve is expected for enzymes following the BET model [19].…”

“…Water adsorption experiments were carried out with both silanized Chromosorb support alone and the same support with immobilized CALB (Figure 2 The water adsorption isotherm shape of Chromosorb with immobilized CALB differs from the sigmoid shape of curves obtained with CALB alone [19], and more generally with isolated proteins [28], which can be modeled by the BET equation.…”

“…For water activities below 0.5, water initially binds to well-defined water-binding sites at the protein surface and then above 0.5, gradually a spanning water network is formed [19].…”

- Global and Local Properties of Mixtures: An Expanded Paradigm for the Study of Mixtures

“…In the latter strategy, conventional MD simulations are carried out, but the water activity is calculated through post-analysis of the simulations. The study of Branco et al (Branco et al 2009) is apparently the only work that explicitly considers water activity as a variable. However, their medium was assumed to be an ideal mixture.…”