FIGURE 4.

Example of the microtiter plate assay for the screening of site-saturation libraries. The first column contains four negative controls (wells 1A–1D), lysate of E. coli BL21 (DE3) Gold carrying pET26b(+) vector without insert, and four positive controls (wells 1E–1H), lysate of E. coli BL21 (DE3) Gold expressing the wild type enzyme. (Left) The picture shows the plate 35 min after addition of the detection solution. (Right: Top) Absorbance values of the same plate measured in a plate reader at 442 nm. (Right: Bottom) Absorbance difference between WT and variants. The screening was performed with α-methylstyrene in presence of tert-butyl hydroperoxide as substrates and vanillin for detection using the standard assay conditions described in Section “Materials and Methods.”