Enhancing promiscuous chemistries of a Schiff-base forming enzyme by divergent evolution

Directed evolution has emerged as a powerful technique for the rapid tailoring of enzymes toward particular synthetic demands, spawning a number of enzymes capable of complex chemical transformations. During random mutagenesis of a protein, changes in fitness must be assayed in order to quantify and understand the relative effect a given mutation has, and the assay employed must be carefully chosen to report directly on the transformation of interest. Here, we describe a series of medium-throughput screening techniques that have been utilized for the evolution and engineering of an artificial carboligase, RA95.5-8, resulting in improvement of catalytic efficiency of a number of promiscuous chemistries. The methods make use of common analytical chemistry equipment and low-cost materials, and may help inspire development of novel screening workflows for related transformations.