Exopolysaccharides consisting of partially de-N-acetylated poly-β-D-(1-6)-N-acetyl-glucosamine (dPNAG) are key structural components of the biofilm extracellular polymeric substance (EPS) of both gram-positive and gram-negative human pathogens. Enzymes that breakdown the biofilm EPS, including glycosidase, DNase and protease enzymes have emerged as promising tools to disrupt existing biofilms. DispersinB (DspB) a β-hexosaminidase from Aggregatibacter actinomycetemcomitans, is the only secreted dPNAG specific polysaccharide hydrolase identified. DspB is categorized as CAZy GH20 familyglycosyl hydrolase enzyme and specifically cleaves dPNAG using a substrate assisted cleavage mechanism. DspB has shown promise for the enzymatic dispersal of bacterial biofilms, DspB shows relatively poor substrate binding affinity. Thus, there is an interest in discovering other dPNAG specific glycosidase as potential biocatalysts for biofilm dispersal. In this study, we have developed a high throughput colorimetric assay to screen for dPNAG glycosidase activity that uses GlcNAc-β-D-(1-6)-GlcNAc-β-D-1-p-nitrophenyl carbamate as a substrate. This assay shows high specificity DspB over other β-hexosaminidase enzymes. The hydrolyzed product, p-nitroaniline, has a distinct absorbance at 410 nm, and can be used to screen for GlcNAc-β-D-(1-6)-GlcNAc hydrolase activity in cell lysates.

Development of a colorimetric assay for high throughput screening of poly-β-D-(1-6)-N-acetyl-glucosamine specific glycosidase activity

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.