Circular permutation of lipase B from Candida antarctica (CALB) offers a new approach for altering and improving enzyme performance; in particular in connection with the enantioselective synthesis and hydrolysis The circular permutation of CALB is presumed to alter the structural integrity of the protein, potentially changing its active site accessibility and flexibility, all factors affecting an enzyme's substrate recognition and turnover. We have generated comprehensive combinatorial circular permutation libraries of CALB and identified functional variants by colony screening for tributyrin hydrolysis. Interestingly, protein variants with relocated termini in or near the enzyme's active site make up a significant portion of functional permutants. Subsequently, in vitro studies of selected CALB variants have identified several candidates with up to 175-fold improved catalytic efficiency (kcat/KM) on various substrates. Data on the substrate specificity and enantioselectivity of these proteins will be presented. Beyond the exploration of the enzyme variants' substrate specificity, we are studying the observed changes in catalysis in the context of enzyme structure, dynamics, and mechanism. Finally, we have utilized our circular permuted catalyst as templates for subsequent rational and combinatorial protein engineering, further exploring sequence space for lipases with novel and improved properties.