(666c) The Impact of Protein Residue-Explicit Covalent Immobilization for Stability Enhancement (PRECISE) on Enzyme Biocatalysis

Biocatalysis is a rapidly expanding multi-billion dollar industry due its renewable, sustainable, and environmental-friendly nature and its stereo-, chemo-, and regio-specificity. However, limitations to enzyme-mediated biocatalysis remain such as the enzyme’s stability and the long-term recoverability/reusability of the enzyme. To overcome this limitation, many enzyme immobilization technologies have been developed; however, these too come with limitations such as enzyme leaching and lack of control over the orientation at which the enzyme is immobilized. Here we present a technique that employs the site-specific incorporation of uniquely reactive unnatural amino acids to control the biocatalysis orientation when covalently attached to the surface through a post-translational modification. This technique, which we refer to as the Protein Residue-Explicit Covalent Immobilization for Stability Enhancement or PRECISE system, is directly compared to traditional covalent immobilization technologies.  Using the PRECISE system we report improved activity and stability under both denaturing and physiological conditions. We also highlight the ease of adoption and high-throughput capability of the PRECISE system.