A combined experimental and computational approach for the rationalization of the catalytic activity of lipase B from Candida antarctica in water-organic solvent mixtures

Background: The addition of organic solvents to an aqueous medium for enzymatic reactions offers several advantages, as they can increase the solubility of substrates but can also lead to enzyme inactivation and/or aggregation.Results: The effect of adding 30% of several water-soluble organic solvents on the catalytic activity of lipase B from Candida antarctica (CalB) was studied and the results showed that the highest activity was obtained with the addition of t-butanol. t-Butanol and acetonitrile were selected and the kinetic parameters, determined to deepen their effect on CalB activity, showed that the addition of acetonitrile improved the enzyme-substrate affinity, while water-t-butanol mixtures led to a more than ninefold increase in k(cat). To rationalize at a molecular level the kinetic results, molecular dynamic simulations were performed. Analysis of the accessibility of the active-site cavity, solvent occupancy in the site and in the oxyanion hole, and the stability of the catalytic triad in the two solvent mixtures, provided insight into their effects on the catalytic properties of CalB.Conclusion: The lower occupancy in the oxyanion hole of water molecules and a shorter residence time in the active site of acetonitrile molecules in the acetonitrile-water mixture contribute to the higher enzyme-substrate affinity found experimentally. Conversely, the higher k(cat) in the t-butanol mixture is explained by the higher stability of the catalytic triad and by an increase in the nucleophilicity of the catalytic serine due to the persistent presence of t-butanol molecules in the active site. (c) 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).