Polymer-Based Protein Engineering Enables Molecular Dissolution of Chymotrypsin in Acetonitrile.

While most effective in aqueous environments, enzymes are also able to catalyze reactions in essentially anhydrous organic media. Enzyme activity in organic solvents is limited as a result of inefficient substrate binding, lack of solubility, and inactivation by hydrophilic anhydrous solvents. With these facts in mind, atom transfer radical polymerization was used to synthesize chymotrypsin-poly(2-(dimethylamino)ethyl methacrylate) (CT-pDMAEMA) conjugates designed to be soluble and active in acetonitrile. CT-pDMAEMA solubility in organic solvents and the rate of CT-pDMAEMA-catalyzed transesterification in acetonitrile were examined at a range of water (0-15 M) and propanol (0.01-5 M) concentrations. The conjugates were soluble at the molecular scale in several organic solvents, exhibited good substrate binding with -acetyl l-phenylalanine thiophenylester ( as low as 17 mM), and had an activity (peak activity 330 μM/min/mg enzyme) many orders of magnitude higher than that of the insoluble native enzyme.