Abstract

Three novel endo-β-N-acetylglucosaminidases (AVUL01, BCAC01, and BFIN01) classified as members of the glucoside hydrolase (GH) family 18 were identified from human fecal samples and then cloned and characterized for their ability to hydrolyze two distinct classes of N-glycans. Endo-β-N-acetylglucosaminidases (ENGases) are known for the hydrolysis of chitin and the N,N′-diacetylchitobiose core of N-linked glycans, depending on the glycan architecture. N-glycans have shown bioactivity as substrates in the human gut microbiome for microbes that encode ENGases, thus demonstrating their ecological relevance in the gut. However, distinct types of N-glycan structures, for example, oligomannosidic or complex, have been shown to enrich different microbes within the human gut. Novel advances in food technology have commercialized animal-derived dietary proteins with oligomannosidic instead of traditionally complex N-glycans using precision fermentation. This indicates that there is an unmet need to identify the classes of N-glycans that gut-derived ENGases act upon to determine whether these novel proteins alter gut ecology. AVUL01, BCAC01, and BFIN01 all demonstrated activity on exclusively oligomannosidic N-glycans from RNase B and bovine lactoferrin; however, they failed to show activity on complex or α-1,3-core fucosylated high-mannose N-glycans derived from fetuin and horseradish peroxidase, respectively. These results suggest that α-1,3 core fucosylation and complex N-glycan architecture inhibit the activity of AVUL01, BCAC01, and BFIN01. Furthermore, BFIN01 performed significantly better than BCAC01, resulting in a greater amount of N-glycans, suggesting that certain ENGases may possess enhanced specificity and kinetics as an evolutionary strategy to compete for resources.