Directed evolution and solid phase enzyme screening

A new digital imaging spectrophotometer and a series of colorimetric solid phase assays have been developed to screen bacterial libraries expressing mutagenized enzymes undergoing directed evolution. This high-throughput solid-phase assay system (known as 'Kcat Technology') can detect less than a 20% difference in enzyme rates within microcolonies grown at a nearly confluent density of 500 colonies per cm(2) on an assay disk Each microcolony is analyzed simultaneously at single-pixel resolution (1.5 megapixels; 75 micron/pixel), requiring less than 100 nanoliters of substrate per measurement, a 1000-fold reduction over conventional liquid phase assays. Here we report the successful identification of variants of Agrobacterium beta-glucosidase (Abg) - a glycosidase with broad substrate specificity that favors cleavage of glucosides over galactosides - by simultaneously assaying two different substrates tagged with spectrally distinct chromogenic reporters. In this study, we have used combinatorial cassette mutagenesis based on the phylogeny of homologous glycosidase genes and known point mutants in Abg to generate a recombinant library of over 10 million enzyme variants involving partial randomization of 13 amino acid residues near the active site. Different enzyme variants that show epimeric specificity either for the hydrolysis of glucoside or galactoside - were identified by solid-phase screening. This solid phase screening technology is compatible with a variety of mutagenesis methods including error-prone mutagenesis, sequential random mutagenesis, and gene shuffling. However, using combinatorial cassette mutagenesis, this greater screening capacity enables one to survey large sequence spaces in which many residues are changed simultaneously. isolating optimized enzymes may be accomplished through recursive cycles of cassette mutagenesis using a GA-based technique, Recursive Ensemble Mutagenesis (REM) - emulating the natural evolution of genes by point mutation and recombination. This technology should lead to the isolation of new enzyme activities that are useful in the synthesis of various substances, including specialty chemicals and pharmaceuticals.