Fig. 2

Screening a human gut metagenomic library for sulfatases. A Coupled assay for sulfatase screening. The substrate consists of a GlcNAc residue linked to the fluorophore 4-methylumbelliferone (4MU). The GlcNAc molecule is modified at carbon 6 with a sulfate group (SNFG notation, GlcNAc, blue square). In a first reaction, a sulfatase expressed from a metagenomic clone removes the C6 sulfate. In a second reaction, an exogenous hexosaminidase (that is inactive on the sulfated substrate) liberates the fluorophore from GlcNAc, generating a fluorescence signal. B Re-screening human gut metagenomic clones for sulfatase activity. Represented are fluorescence values for the 30 h timepoint. Values above the mean + 6σ (black line) were considered ‘hits’. Control lysates from clones carrying an empty pCC1 fosmid (grey circles) were assayed along with the metagenomic clones (blue circles) from the primary screen