Screening Fosmid Library for Carboxylesterase and Lipase Activities

The metagenomic libraries were screened for carboxylesterase and lipase activity as follows. The fosmid library was grown on LB agar plates containing 12.5 μg mL⁻¹ chloramphenicol at 37°C overnight to yield single colonies. Then, 3,456 clones were arrayed in 9- by 384-well microtiter plates and cultivated at 37°C in LB medium supplemented with 12.5 μg mL⁻¹ chloramphenicol. Those original microtiter plates were stored at –80°C after the addition of glycerol, at a final concentration of 20% (vol/vol). For screening clone libraries, 384-pin replicators were used to print clones onto the surface of large LB agar square plates (245 mm by 245 mm) containing 12.5 μg mL⁻¹ chloramphenicol, and 2 mL liter⁻¹ fosmid autoinduction solution (Epicentre), and each plate contained 0.3% (vol/vol) tributyrin (Sigma-Aldrich, Gillingham, United Kingdom) as described previously (27 (link)). After an initial overnight growth at 37°C, the LB agar plates were incubated for 48 h at 37, 50, or 70°C. Positive hits were confirmed by retesting the corresponding fosmid clones taken from the original microtiter plate.