Spectroscopic Kinetic Monitoring and Molecular Dynamics Simulations of Biocatalytic Ester Hydrolysis in Non-Aqueous Solvent

The use of enzymes as catalysts is becoming an increasingly important tool in chemical synthesis, given the mild conditions and high chemoselectivity that can be achieved through enzyme-catalyzed reactions. However, a major limitation in the use of enzymatic biocatalysis is the degradation of enzyme structure and activity in non-aqueous media. Lipases are a subclass of esterases found in most, if not all, living organisms that break ester bonds in lipids. In this study, we explored the effects of various concentrations of non-aqueous organic solvents on pancreatic lipase activity and analyzed the relationships between different properties of solvents and the kinetics of enzymatic hydrolysis through spectroscopic monitoring of a synthetic colorimetric substrate, 4-nitrophenyl acetate. The influence of non-aqueous solvent environments on protein stability was further explored with molecular dynamics (MD) simulations on a 1 nanosecond timescale. Our results suggest a general trend of decreasing enzymatic activity with increasing concentrations of non-aqueous solvent; however, lipase activity in low concentrations of methanol, specifically 5% methanol, was 28% higher than lipase activity in water. Lipase activity in methanol also displayed the greatest rate of hydrolysis of the substrate compared to all other non-aqueous solvents. Interestingly, our MD simulations showed that the conformational state and stability of lipase in methanol is similar to that of the enzyme in water. Lipase activity works the best in 5% methanol which can be used in industry for chemical synthesis and enzyme-catalyzed reactions.