We then further optimized GNCA4-WT 28 using the FuncLib server 21 on the basis of the screening of a small ultra-focused library randomizing 11 positions, that included variants with multiple active-site mutations at the top of the stability ranking predicted by FuncLib. This resulted in a Kemp eliminase with a catalytic efficiency of ~2•10 4 M -1 s -1 , a catalytic rate constant of ~100 8 s -1 and a denaturation temperature of 76 o C, as determined by scanning calorimetry. We shall refer to this variant as GNCA4-12, in line with our prior work. Finally, GNCA4-12 was further enhanced on the basis of the inclusion of an extra polypeptide segment at the carboxyl terminus (Figure ). The rationale behind this approach was that the extra polypeptide segment could generate new interactions at the adjacent de novo active site and that these interactions could perhaps be tapped for catalysis enhancement. In order to do so, a library including all combinations of the 20 amino acids at three positions in the extra segment was prepared. This combinatorial library spanned 8000 variants, of which ~800 were screened for Kemp elimination activity. The best variant thus obtained had a catalytic efficiency of ~2•10 5 M -1 s -1 , a catalytic rate constant of ~600 s -1 and a denaturation temperature of 80 o C. We shall refer to this variant simply as V4, following the terminology used in the original work. Note that the approach based on the inclusion of an additional polypeptide segment does not rely on features specific to Kemp elimination or to the protein scaffolds we use for de novo enzyme engineering. It appears thus reasonable that the approach is general to a substantial extent, although additional studies on other enzyme systems will be required to confirm this statement. This Kemp eliminase variant, V4, resulting from previous studies described above, provides the starting point for the current work. It is already highly optimized in terms of both stability and catalysis. Its denaturation temperature value, 80 o C, is, for instance, much higher than that for the prototypical TEM1 b-lactamase (55 o C). Regarding de novo Kemp eliminase catalysis on this scaffold, the catalytic parameters for V4 (kcat ~600 s -1 and kcat/KM ~2•10 5 M -1 s -1 ) are similar to those of the best proton-abstraction Kemp eliminase reported to date (HG3.17 kcat = 700 s -1 , kcat/KM = 2.3•10 5 M -1 s -1 32 ), which was the result of 17 rounds of directed evolution starting with a rationally designed background with a low activity. We note here that a recent even more efficient Kemp eliminase has been designed with a catalytic rate constant of ~3600 s -1 ; however, this enzyme does not use a conventional proton-abstraction mechanism and is a redox-mediated enzyme based on using myoglobin as a starting point. It is clear overall that further optimization of V4 appears a priori to be a challenging prospect. This challenge is why we chose the V4 variant as an engineering target to stress-test our approach. Experiments performed as part of the current study and aimed at further enhancing the V4 variant are described below.