β-Mannan degradation by gut bacteria - Characterization of β-mannanases from families GH5 and GH26

The human gut flora is important for our well-being. The gut bacteria are able to degrade and metabolize complex carbohydrates. Examples of such carbohydrates are β-mannans. β-Mannans consist of a backbone of β-1,4-linked mannose units and are present in e.g. the endosperm of legumes such as guar or carob. The composition of the β-mannan varies with the plant source. Carob and guar β-mannans are substituted with α-1,6-linked galactose units and are soluble in water. They make a viscous solution that is used e.g. as food thickener. It is known that guar gum galactomannan can be fermented by the human gut flora. The main enzymes that hydrolyze β-mannan backbones are called β-mannanases. β-Mannanases have been studied and characterized from a range of environments. However, β-mannanases have yet not been studied from the gut flora to any significant extent. This thesis focuses on β-mannanases from families GH5 and GH26 from the gut bacteria Bacteroides and Bifidobacterium. In Paper I, we have studied the effect of galactomannan on metabolic markers and four bacterial genera in rats fed with guar gum. Guar gum of different viscosities was tested. We found that Bifidobacterium counts were increased when the rats were fed guar gum, regardless of viscosity, while the number of Bacteroides was not different from the control. In Papers II-IV we characterized four enzymes from these genera possibly involved in the degradation of guar gum and other mannans. We characterized BaMan26A, a GH26 β-mannanase from Bifidobacterium adolescentis in Paper II and a GH5 β-mannanase, BlMan5_8, from Bifidobacterium animalis subsp. lactis in Paper IV. In Paper III we characterized two GH26 β-mannanases, BoMan26A and BoMan26B, from Bacteroides ovatus that are encoded by a polysaccharide utilization locus. A crystal structure of BoMan26A displayed the (α/β)8 fold of clan GH-A enzymes and a substrate binding cleft. The four β-mannanases were found to vary in product profile and fine-tuned substrate specificity within the group of β-mannans. While BlMan5_8 produced oligosaccharides of varying length from β-mannan, BoMan26A produced almost exclusively mannobiose from β-mannan. BoMan26B produced mainly mannobiose, while BaMan26A produced mannotriose as a major product. BlMan5_8 is predicted to be secreted, while BoMan26B and BaMan26A appear to be anchored to the cell. BaMan26A is predicted to be located in the periplasmic space. This work gives further insight in the molecular details of β-mannan catabolism in the gut