“…Fifty microliter of the whole cell suspension were added to 150 mL of the assay solution (140 mL HEPES, 1 mM, pH 8.2, 20 mg/mL phenol red supplemented with 10 mL of a mixture of 100 mM haloalkanes 1,2-dibromoethane, 1-iodopropane, 1-bromobutane, 1-iodobutane, and 4-bromobutyronitrile in acetonitrile). All five substrates are known to be accepted by the HLD and using a mixture increases the chance to find desired active variants (Fibinger et al, 2015). The absorption at 430 and 560 nm was measured at the beginning and after 24 h of incubation at 37 C. The phenol red indicator color change to yellow, indicated by a change in the ratio of both absorption values, was used for signaling the release of protons and subsequent catalytic conversion of the haloalkanes to the corresponding alcohols.…”