Our goal in this work is to use NMR-guided directed evolution combined with computationally focused library screening to optimize the V4 Kemp eliminase (the best Kemp eliminase from our previous work ). As our starting point, we initially aimed at obtaining NMR chemical shift perturbations (CSP) with this variant. However, the inclusion of the extra polypeptide segment (Figure ) in this variant makes it prone to slowly aggregate in solution, which makes V4 unsuitable for long-time NMR experiments. As a substitute for this, we performed NMR experiments and subsequent computational optimization using the GNCA4-12 Kemp eliminase, under the reasonable assumption that the hotspots thus determined could also be used for V4 optimization due to the sequence similarity between the two enzymes (which only differ in the presence of the additional segment in V4). Following previous work in the field, we used 5(6)-nitrobenzotriazole (Figure ) as an analogue of the transition state for Kemp elimination.