Initially, the best Kemp eliminase obtained through this approach 28 involved a single W229D mutation in the GNCAMP scaffold, i.e., in the protein encoded by the most probabilistic sequence at the GNCA ancestral node (last common ancestor of various Gram-negative bacteria). This Kemp eliminase had a catalytic efficiency of 45 M -1 s -1 although its catalytic rate constant, kcat, could not be accurately determined. Further improvements in activity were achieved by introducing an additional mutation (F290W) at the de novo active site and by screening several alternative reconstructions at the GNCA node as scaffolds for engineering. This Kemp eliminase will henceforth be referred to as GNCA4-WT, as it provided the baseline variant for our subsequent optimization effort. It is important to note that the same design approach did not generate Kemp eliminase activity when implemented on 10 different modern b-lactamases of moderate stability that were conformationally rigid. Introduction of the additional F290W mutation let us reach a catalytic efficiency of ~5•10 3 M -1 s -1 with a catalytic rate constant as high as ~10 s -1 for Kemp elimination by GNCA4-WT. It is also worth noting that the natural activity of b-lactamases is the degradation of b-lactam antibiotics, such as penicillin. Our design approach generated a de novo active site that is both chemically distinct and spatially separated from the natural antibiotic degradation active site.