Abstract 1297 Combined rational and combinatorial refinement of the substrate specificity and enzyme reactivity of a biocatalytic esterase

Esterases are essential enzymes involved in numerous cellular functions such as recycling proteins and mediating signaling pathways. Esterases are also utilized as biocatalysts in the industrial manufacturing of pharmaceuticals and detergents. BS2, an esterase from Bacillus subtilis, is a highly versatile esterase with the unusual ability to hydrolyze esters of tertiary alcohols, which are not hydrolyzed by human esterases. Combining this emergent reactivity for tertiary esters with the ability to split BS2 into reconstituting half sections has made BS2 into a tool for studying protein-protein interactions in human cells. In this study, we expanded the biocatalytic properties and esterase reactivity of BS2 by further expanding its substrate reactivity toward diverse ester substrates. Using a comprehensive alanine scan of its binding pocket and a diverse library of fluorogenic ester substrates, we identified unique substrate reactivity within BS2 and correlated this unique reactivity with multiple binding pocket hotspots that regulate the substrate specificity of BS2. Randomized combinatorial libraries were then synthesized for two binding pocket residues whose alanine variants showed promising shifts in substrate reactivity and proper library diversity was confirmed by sequence analysis. Random libraries were then screened via pooled high-throughput fluorescence assays against a subset of orthogonal ester substrates and the increased activity of the resulting variants was confirmed by complete enzyme kinetic measurements against purified final proteins. Matched BS2 variants and ester substrates will make valuable tools for sensitive visualization of protein-protein interactions and as novel biocatalysts.