Enzyme Microheterogeneous Hydration and Stabilization in Supercritical Carbon Dioxide Rodrigo L. Silveira, † Julian Martínez, ‡ Munir S. Skaf, † and Leandro Martínez* ,§ † Institute of Chemistry, and ‡ Faculty of Food Engineering, State University of Campinas, Campinas, SP, Brazil § Institute of Physics of Sã o Carlos, University of Sã o Paulo, Sã o Carlos, SP, Brazil * S Supporting Information ABSTRACT: Supercritical carbon dioxide is a promising green-chemistry solvent for many enzyme-catalyzed chemical reactions, yet the striking stability of some enzymes in such unconventional environments is not well understood. Here, we investigate the stabilization of the Candida antarctica Lipase B (CALB) in supercritical carbon dioxide-water biphasic systems using molecular dynamics simulations. The preserva- tion of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating its catalytic core. Strikingly, water and supercritical carbon dioxide cover the protein surface quite heterogeneously. In the first solvation layer, the hydrophilic residues at the surface of the protein are able to pin down patches of water, whereas carbon dioxide solvates preferentially hydrophobic surface residues. In the outer solvation shells, water molecules tend to cluster predominantly on top of the larger water patches of the first solvation layer instead of spreading evenly around the remainder of the protein surface. For CALB, this exposes the substrate-binding region of the enzyme to carbon dioxide, possibly facilitating diffusion of nonpolar substrates into the catalytic funnel. Therefore, by means of microheterogeneous solvation, enhanced accessibility of hydrophobic substrates to the active site can be achieved, while preserving the functional structure of the enzyme. Our results provide a molecular picture on the nature of the stability of proteins in nonaqueous media. 1. INTRODUCTION Supercritical carbon dioxide (scCO 2 ) is a promising solvent for green chemistry, as it is nontoxic, nonflammable, can be inexpensively produced by many industrial processes, and is able to solvate both hydrophobic and hydrophilic solutes. 1 Furthermore, solutes can be readily recovered from scCO 2 by depressurization. Synthetic chemistry in scCO 2 is, therefore, becoming increasingly important in industrial applications. 2 Classical organic chemistry can be adapted to scCO 2 usually with the sole condition that reactants and products do not react directly with CO 2 . Reactions can be performed with solid catalysts, for instance, by simply pumping reactants and products dissolved in scCO 2 through a porous support for the catalyst. 2 However, some reactions are hardly transferable to scCO 2 as some of their constituents either react with CO 2 or depend on the aqueous medium for activity. Among the latter, enzyme catalyzed reactions are most challenging since, although proteins may be stable in many uncommon media, virtually any protein evolved to preserve its fold in highly polar aqueous conditions, which hardly mimic scCO 2 environments. Therefore, it is fascinating that naturally occurring (not engineered) enzymes are progressively being studied and used for catalysis of organic reactions in scCO 2 , thus preserving their fold in scCO 2 to the point of maintaining, and even, increasing, their catalytic activity. 3,4 How can enzymes, or proteins in general, having evolved to function in aqueous solutions, preserve their structure and activity in such nonstandard environments is a question of fundamental importance for understanding the nature of protein folding, stability, solvation, and interactions, and has practical implications in protein design for green and sustainable chemistry. Here we use molecular dynamics (MD) computer simulations to study molecular aspects of the stability of the Candida antarctica Lipase B (CALB) in scCO 2 . This and similar lipases have been intensively used in the food and cosmetic industry for enantioselective hydrolysis of esters, and their stability in scCO 2 has been largely reported, 3-7 being one of the most promising enzymes for catalysis in scCO 2 . As these enzymes catalyze the hydrolysis of triglycerides, which are hydrophobic substrates, the scale-up strategy for their application depends on their dispersion in nonpolar solvents. 2,5 However, scCO 2 has been shown to be a viable alternative which increases by almost 2 orders of magnitude the activity of lipases relative to hexane, for example. 8 Additionally, these enzymes increase their activity upon binding to oil-water interfaces, a remarkable property that is related to a large Received: February 22, 2012 Revised: April 4, 2012 Published: April 12, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 5671 dx.doi.org/10.1021/jp3017858 | J. Phys. Chem. B 2012, 116, 5671-5678