This allows for an extended substrate concentration range, but at the slight expense of catalytic efficiency. Such studies in the presence of 5% acetonitrile were performed at pH 7 for all the 20 top variants of the FuncLib ranking (Table ) and, as a function of pH for the 4 best variants. The corresponding profiles of catalytic efficiency and turnover vs. pH are compared with those for our background protein, GNCA4-WT in Figures and. These data confirm an enhancement of catalysis over background of up to about one order of magnitude, in particular in the kcat value. It is to be noted, nevertheless that while the addition of 5% acetonitrile has the crucial advantage of increasing the solubility of the substrate for the Kemp elimination reaction, thus expanding the experimental concentration range and allowing for more accurate determination of catalytic parameters, the presence of such a small amount of acetonitrile has a small detrimental effect on catalysis (a decrease of about 2-fold), likely in part through a general solvent effect.