Metagenomic Screening for Lipase-Producing Clones

Purified DNA samples were size-separated on a 0.7% low melting point (LMP) agarose gel in TAE buffer at 20 V/cm. DNA bands from 20 to 40 kb were excised from the gel and extracted by the phenol method [57 ]. A metagenomic DNA library was constructed using the CopyControl Fosmid Library production kit, according to the manufacturer's protocol (Epicentre Biotechnologies, Madison, WI, USA). For activity screening, the transformed cells were plated onto modified Luria-Bertani (LB) agar plates (5 g/L peptone, 3 g/L yeast extract, 13 g/L bacteriological agar, 10 g/L gum arabic) containing 1% (v/v) emulsified tributyrin as substrate. Cells were grown at 37°C for four days and transformants with clear halos around individual colonies were chosen as possible lipase/esterase producing clones. The selected clones were transferred to 96-well microtiter plates with Terrific Broth and subjected to a second screening for lipolytic activity on the modified LB agar except that 1% (v/v) tricaprylin was used instead of tributyrin. Tricaprylin positive clones were transferred to 96-well microtiter plates and stored. True lipase producing clones were identified amongst the tricaprylin active clones by screening on modified LB agar containing 1% (v/v) triolein. Three clones that showed the strongest lipase activity (FosC12, FosE6 and FosH10) were further analyzed.