Thermoinactivation of diisopropylfluorophosphatase-containing polyurethane polymers

The thermoinactivation of native diisopropylfluorophosphatase (DFPase, EC 3.8.2.1) is highly calcium dependent, first-order kinetic. Deactivation is coupled with a simultaneous reduction in beta-sheet content. We report herein our attempts to enhance the thermostability of DFPase by irreversibly incorporating the enzyme into polyurethane polymers. Immobilized DFPase has biphasic deactivation kinetics. Our data demonstrate that the initial rapid deactivation of immobilized DFPase leads to the formation of a hyperstable and still active form of enzyme. Like native DFPase, DFPase-containing polyurethanes exhibit a calcium-dependent thermostability. Since bioplastics cannot be analyzed by spectroscopy, the structural mechanisms involved in thermoinactivation of immobilized DFPase were determined using PEG-modified DFPase. The thermoinactivation profile of highly modified DFPase mirrors the stepwise deactivation pattern of bioplastics. Spectroscopic studies enable a structural analysis of the hyperstable intermediate.