(169b) Kinetic Stabilization of An Enzyme by Insertion Into a Thermophilic Host Protein

Insufficient stability of enzymes is a fundamental problem that restricts their application in many areas. Enhanced stability achieved by conventional stabilization approaches frequently sacrifices catalytic activity. We show that domain insertion into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) can improve kinetic stability of two unrelated model enzymes. The entire enzyme domains were inserted into PfMBP and a mesophilic homologue from Escherichia coli (EcMBP) at rationally selected sites, after which expression levels, thermodynamic and kinetic stability of resulting protein complexes were examined. We provide evidence that insertion into several locations of PfMBP improved kinetic, but not necessarily thermodynamic, stability of the enzyme domains without any compromise in enzyme activity. However, thermodynamic and kinetic stability of the enzyme domain was significantly compromised upon insertional fusion to EcMBP. Overall, the observed kinetic stabilization of two unrelated model enzymes by insertion into PfMBP suggests the high promise of applicability of our novel approach into a wide range of proteins. Also, we provide evidence that the nature of a host protein determined the stability of a guest inserted protein domain.