(408f) Evaluating the Effect of Attachment Orientation On the Activity and Stability of Immobilized Enzymes

Due to the overwhelming demand for environmentally friendly manufacturing processes, the multibillion dollar biocatalysis industry is rapidly expanding.  Biocatalytic systems enable stereo-, chemo-, and regio-specificity in chemical manufacturing, reducing the production of wasteful byproducts.   This specificity is particularly valuable in industries such as the pharmaceutical industry, where removal of chemically similar but physiologically harmful waste products is essential.  Traditional biocatalytic processes suffer from limitations in enzyme stability, leaching, recoverability, and reusability.  Given the high cost of enzyme production, these limitations significantly hinder the cost-effectiveness of biocatalysis for industrial applications.  These traditional processes of enzyme immobilization such as adsorption, cross-linking, and entrapment provide some improvements to stability, recoverability, and reusability but suffer from enzyme leaching, complicated or toxic conjugation procedures, and a lack of attachment orientation control.  The limited number of canonical amino acids presents an additional obstacle to site-specific attachment orientation control as it is difficult to target a single attachment location.  Here, we build upon recent advancements in unnatural amino acid incorporation to demonstrate a biocompatible, bioorthogonal, and covalent enzyme immobilization process that improves protein stability and enables attachment orientation control.  This system, which we refer to as the Protein Residue-Explicit Covalent Immobilization for Stability Enhancement or PRECISE system, permits the covalent attachment of enzymes at potentially any specific residue onto a surface.  Using this process, we report the effect of attachment orientation on enzyme activity, durability, and stability in harsh and mild environments.