Fluorescence-based pH-shift assay with wide application scope for high-throughput determination of enzymatic activity in enzyme mining and engineering

A number of enzymes important for biocatalyst development or as drug targets are associated with a pH shift during their catalytic reaction, owing to the concommitant release or uptake of protons. Here, we show that an enzyme assay developed using the fluorescent pH indicator HPTS can be adapted for reliable and continuous activity determination of representative enzymes from multiple EC classes that operate in the viable pH range 5.5–8.5, using ratiometric measurement (F₄₈₅/F₄₀₅). Kinetic measurements obtained with this method closely match literature values determined using other assay types. Further, the assay was employed to screen variants of transketolase from Geobacillus stearothermophilus (TKgst) aimed at engineering substrate promiscuity and remote enantioselectivity for 3-hydroxyaldehydes. The fluorescence-based assay displayed 70-fold improved sensitivity in comparison to an absorption-based assay for transketolase screening, with a limit of detection of 0.044 mM and Z-factor of 0.52. Double-site mutagenesis at the G264 and S385 positions yielded variants with 5–15-fold increased activity on the tested 3-hydroxyaldehydes compared to the TKgst (L382F) base variant. Although the directed evolution engineering strategy did not achieve significant remote enantioselectivity in this first round of mutagenesis, the simple fluorescence-based pH-shift assay was shown to be useful as a versatile primary high-throughput screen for in vitro enzyme engineering.