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Circular permutation of enzymes offers an alternative protein engineering strategy forchanging the substrate specificity, as well as improving enantioselectivity and catalyticperformance of biocatalysts. In previous work on lipase B from Candida antarctica (CALB),we demonstrated that the reorganization of the polypeptide sequence results in significant rateenhancements. These functional changes were linked to structural rearrangements alteringactive site accessibility and backbone flexibility. To more broadly explore the potential functional benefits of circular permutatedenzymes, we have now expanded our engineering studies to include two new biocatalystswith distinct folds: the xylanase from Bacillus circulans (BcX) and old yellow enzyme fromSaccharomyces cerevisiae (OYE). BcX is an endo-acting glycosyl hydrolase with a betajellyrollfold while OYE is a member of the flavin-dependent oxidoreductase family with analpha/beta barrel topology. The results from our random circular permutation experimentsand the structural and functional characterization of selected enzyme variants will bepresented. In the case of OYE, the effects of active site reorganization are not limited tosubstrate specificity and enantioselectivity but can potentially extend to changes in chemistry, due to the remodeling of the flavin cofactor environment. Besides highlighting the method’ ability for creating novel biocatalysts, our new studies also demonstrates that not all enzymesare created equal when it comes to engineering by circular permutation.