(474f) Stabilization of An Enzyme by Domain Fusion to a Thermophilic Protein

We report a novel method for stabilization of target enzyme domains without any modification of their primary sequence. Our method employs domain insertion of a target enzyme into a thermophilic scaffold protein. Insertion of a model target enzyme, exoinulinase (EI), into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability of the EI domain without any compromise in its activity. Insertion of TEM-1 beta lactamase (BLA) at this same site in PfMBP stabilized BLA without altering its substrate specificity, suggesting that the described method can potentially be applied to a wide range of proteins. The model study suggested that the improved kinetic stability should be due to a raised kinetic barrier for irreversible alteration of unfolded intermediates to completely inactivated species.