Linear and branched β-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288^T and their roles in cooperation with gut bacteria

Abstract

β-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288 T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for β-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo-β-1-3 linkages while BuGH30 can digest endo-β-1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting β-glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain β-1-6 linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288 T is not only capable of utilizing β-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.

Human gut Bacteroides uniformis utilizes mixed linked β- glucans via an alternative strategy

“…As both strains ( B. uniformis JCM 5828 and JCM 13288 T ) showed growth on mixed linkage BβG, we explored the possible role of Bu GH3 MLG in utilizing oligosaccharides generated by Bu GH16. The Bu GH16 of the B. uniformis JCM 13288 T showed 100% homology to one protein characterized from B. uniformis JCM 5828 (Singh, et al ., 2020). A study by Dejean, et al .…”

“…The growth curve pattern of the B. uniformis strains was monitored on 1% barley β glucan (BβG). Both strains were initially cultured in 5 ml GAM and once growth was seen, we performed growth curve experiment in minimal medium containing 1% BβG as per our previously used protocol (Singh, et al ., 2020).…”

“…We recently identified two β glucans utilization loci in the B. uniformis JCM 13288 T and observed that PUL 36 (laminarin) associated Bu GH3 LM showed lower activity with gentiobiose (β-1, 6-linked disaccharide) and cellobiose (β1-4 linked disaccharide) (Singh, et al ., 2020). It was also observed that Bu GH16 present within the LUL can digest mixed linkage (β1-4/ β1-3) glucan, so-called BβG (Dejean, et al ., 2020).…”

“…Both fractions were then freeze-dried. MALDI-TOF and 13 C-DEPT NMR were performed as carried out previously (Singh, et al ., 2020). MestreNova software was used for 13 C-DEPT NMR data processing.…”

“…Recently, our lab (Singh, et al ., 2020) and others (Dejean, et al ., 2020) have identified and dissected the molecular mechanism of the laminarin utilization locus (LUL) in the Bacteroides uniformis JCM13288 T and Bacteroides uniformis JCM5828 (ATCC 8492) respectively. These studies have established that LUL associated glycoside hydrolase-16 (GH16) can cleave mixed linkage ( β 1-4/ β 1-3) glucan of the barley, but GH3 of the same PUL did not show any significant activity for digesting β 1-4/ β 1-3 linked oligosaccharides.…”