As mentioned before, FuncLib combines phylogenetic analysis and Rosetta calculations to suggest multiple mutations that generate stabilizing interacting networks at the active site. Indeed, the denaturation temperatures of the top 20 variants, as determined by differential scanning calorimetry demonstrate that all enzymes are stable, and two variants even appear to be somewhat more stable than the background. (Table ). This confirms that, despite the substantial number of mutations introduced, the Funclib predictions avoid substantial protein destabilization. This should be compared with the top ten variants derived from the random library screening (Table ) which, in some cases, display substantially diminished denaturation temperatures. a Catalytic parameters were determined at pH 7 in the presence of 5% acetonitrile and the His-tag, from fits of the Michaelis-Menten equation to the experimental profiles of rate vs. substrate concentration. The use of 5% acetonitrile extends the experimentally available substrate concentration range, but has a slightly detrimental effect on activity (see Figure ). This explains the difference between the value given in this table for the "wild type" protein and that given in Table . Michaelis plots for variants GNCA4-4 and GNCA4-6 were almost linear, even with the extended substrate concentration range allowed by the addition of 5% acetonitrile. This explains the large uncertainty associated to the determination of kcat and Km for these variants, specifically. Note that the number following "GNCA" in the variant column corresponds to the ranking of the FuncLib Prediction, based on the Rosetta score, as provided in the Supplementary Data and in Table . The GNCA4-WT baseline variant is referred to here as the "wild-type"