To this end, iterative rounds of site saturation mutagenesis were performed using NNK degenerate codons, targeting residues within close proximity to the Zn 2+ cofactor and the substrate binding pocket. Beneficial diversity identified during rounds of evolution was combined by DNA shuffling. The aforementioned spectrophotometric assay was used to evaluate individual variants as crude cell lysates arrayed in 96-well plates, using 50 mM 2 as the substrate and 1% NH2OH (pH 7) as the reaction medium. Throughout evolution, we identified improved variants with kinetic profiles consistent with rapid initial accumulation of 4 beyond the equilibrium position followed by slower redistribution of products to equilibrium (Fig. ).