Alginate lyases from human gut Bacteroides

Microbial community of the human gut, the so-called microbiota, consists of hundreds to thousands of species. Among these, Bacteroides species have evolved specific strategies for the breakdown of carbohydrates. Previous studies have in fact shown the presence of a repertoire of genes involved in the metabolism of host-derived and diet-derived polysaccharides. Bacteroides species appear able to degrade alginate, a polysaccharide extracted from the cell wall of brown seaweeds that finds a broad use as a food ingredient. However, details of its breakdown and utilization by Bacteroides species remain to be fully investigated. On this basis, Bacteroides cellulosilyticus, Bacteroides xylanisolvens, Bacteroides fragilis, Bacteroides ovatus and Bacteroides vulgatus were chosen to study the breakdown of alginate in vivo by measuring their growth in three different media containing low molecular weight alginate (40 kDa) as the only source of sugars. Despite the first screening revealed the potential ability of two Bacteroides species to metabolize the polysaccharide, further experiments did not confirm the previous measurements, excluding the possibility to use them for more detailed studies. A predicted alginate lyase from Bacteroides xylanisolvens was overexpressed in E. coli and purified to test the breakdown of low molecular weight alginate (40 kDa). However, no activity was measured in presence of the polymer. Further screenings on other putative substrates did not lead to the characterization of the protein. Four potential alginate lyases belonging to Polysaccharide Lyase family 15 (PL15), which specifically includes alginate lyases, were tested on low molecular weight alginate (40 kDa). Despite they turned out to be inactive on the substrate, three of them, unlike what expected, showed a lytic activity on heparin, a polysaccharide stored in the granules of mast cells. Thin Layer Chromatography (TLC), enzyme kinetics and Differential Scanning Calorimetry (DSC) were done to further characterize the proteins. This is one of the first examples of lyases classified as members of PL15 that catalyze the breakdown of heparin.