Exploration of the active-site hotspot set for Kemp elimination activity. Effects of mutations at the active site on enzyme activity may be related with very specific interactions of the mutated side chains. Therefore, we deemed convenient to perform screening of saturation mutagenesis libraries at the three positions of the active-site set, 48, 50 and 225 (Figure ). We did not find any significant enhancement in Kemp eliminase activity at either of the three positions, a result which supports that the active site region has been already optimized to a large extent in previous engineering efforts.