We previously used a minimalist approach (based on 1-2 mutations) to generate a completely new active site for Kemp elimination in ancestral β-lactamase scaffolds. We first attempted to enhance the activity level of our best de novo Kemp eliminase through using standard library screening procedures. A library of variants with random mutations and average mutational load of 3-5 mutations was prepared and 522 clones were tested, as we have described in the Materials and Methods. The corresponding plot of activity relative to background vs. clone ranking is shown in Figure . Of these clones, about 300 showed greatly diminished activity levels, suggesting that the encoded proteins may have failed to fold properly. We randomly chose 4 of these clones for protein preparation and, as expected, we found essentially no soluble protein. We also prepared the proteins for the top 10 clones shown in Figure . In the primary screening, these clones showed activity levels about twice or higher that of the background variant. However, of these clones, only one was confirmed as a real positive in secondary screening carried out with the purified protein (Table ). The corresponding variant included 6 mutations, with catalytic parameters that were only about two-fold higher than those of the background enzyme. a The GNCA4-WT β-lactamase, which is used as the baseline for our study, is referred to in this table as "wild-type" ("GNCA4-WT"). Note that this data for the "wild type" was measured without a His-tag in ref. , which accounts for the small difference with the data given in Table (taken also from ref. ). Kinetic measurements were performed as described in the Methodology section, and kcat, KM, and kcat/KM values are provided in s -1 , mM, and M -1 s -1 , respectively. ∆G ‡ exp and ∆G ‡ calc denote the experimental and calculated activation free energies for these enzymes, in kcal•mol -1 . ∆G ‡ exp was derived from kcat using transition state theory, and ∆G ‡ calc is shown as averages and standard error of the mean over thirty individual EVB trajectories per system. All the values in this table were measured at pH 7 with no acetonitrile (other than the small amount coming from the substrate stock solution). All kinetic measurements were performed at 25 °C.