FIG 4.

Sialidase activity of culture supernatants containing NanI, NanJ, or NanH using different substrates. Sialidase activity assays were performed using the EnzyChrom neuraminidase assay kit. For each substrate, the sialidase activity was determined using supernatants removed from five independent cultures (wild-type CN3718 expresses all three sialidase genes; BMC205 has null mutations in all three sialidase genes [17]; ENanJ expresses only the nanJ gene, ENanI expresses only the nanI gene, and ENanH expresses only the nanH gene) grown for 16 h at 37°C in TH broth. Substrates assayed included 3′ sialyllactose (2,3-linkage), 6′ sialyl-d-lactose (2,6-linkage), and colominic acid sodium (2,8-linkage). (A) Sialidase activity (after subtraction of the background using BMC205 results) was determined using three sialic acid linkage substrates. (B) Percent contribution of each sialidase to wild-type supernatant sialic acid release from each substrate (wild-type value equals 100%). All experiments were repeated at least three times.