Cytochrome c peroxidase (I) from Saccharomyces cerevisiae was subjected to directed mol. evolution to generate mutants with increased activity against ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. Using a combination of DNA shuffling and satn. mutagenesis, mutants were isolated which possessed >20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small org. mol., guaiacol, were not significantly affected. Mutations at residues Asp-224 and Asp-217 were responsible for this increase in activity. These 2 residues are located on the surface of the protein and not in the direct vicinity of the distal cavity of the peroxidase, where small org. substrates are believed to be oxidized. Mutations at position Asp-224 also lead to an increased amt. of the active holoenzyme expressed in Escherichia coli, favoring the selection of these mutants in the employed colony screen. Possible explanations for the effect of the mutations on the in vitro activity of I as well as the increased amt. of holoenzyme were discussed. (c) 2001 Academic Press. [on SciFinder (R)]