pH Sensing Agar Plate Assays for Esterolytic Enzyme Activity

Lipases and esterases are some of the most extensively used enzymes for biotransformations (1 –3 ). This is in part due to their widely applicable chemistry, often exceptional regio- and stereo-selectivity, lack of required cofactors and their functionality in organic as well as aqueous media. These enzymes catalyze the hydrolysis of a vast array of ester substrates. However, the process of identifying a native enzyme with the desired activity against a target substrate can be an arduous task. Directed evolution is a facile strategy for engineering substrate specificity and other properties of a candidate enzyme via beneficial mutation of key residues (4 ). The rate limiting step in directed evolution experiments is typically the development of a functional assay for the targeted activity. Owing to the significant investment of time and effort required to develop useful enzyme screens, general assays that may be applied to more than one enzyme or more than one substrate are of particular importance in the field of directed evolution.