Exploring beta-mannan acting glycosidase families in hydrolysis and mannosyl transfer

The thesis revolves around the study of glycoside hydrolases (GH), specifically GH5 β-mannanases and GH36 α-galactosidase (I-III), as well as a GH130 mannoside phosphorylase (I, IV). These enzymes are investigated for their actions on and conversion of hemicellulosic β-mannan, including food-grade galactomannan and softwood galactoglucomannan, along with manno-oligosaccharides (MOS) derived from such polymers. The focus lies on exploring the transferase activity, specifically transglycosylation, of retaining GHs, elucidating the molecular and structural mechanisms governing these reactions, and assessing their potential applications in synthesizing novel glycosides like MOS and allyl glycosides from renewable glycans (II, III).
Paper I explores the utilization of β-mannan-oligosaccharides (MOS/GMOSs) by Roseburia hominis, and its interactions with Bifidobacterium adolescentis, revealing differential utilization patterns, cross-feeding of acetate, and potential synergistic roles of specific enzymes shown to be upregulated in the presence of MOS/GMOS by R. hominis. In Paper II, A novel double mutant (R171K/E205D) of the catalytic module (CM) of the fam- ily GH5 Trichoderma reesei β-mannanase (TrMan5A) showed enhanced transglycosylation capacity, particularly with mannotetraose and allyl alcohol, surpassing wild-type TrMan5A and offering potential applications in the enzymatic synthesis of novel biomaterials and glycopolymers through allyl glycoside production. The +2 subsite of MeMan5A, a GH5 β-mannanase from the blue mussel, was studied by substituting two tryptophans with alanines in Paper III. These substitutions impaired MeMan5A’s hydrolytic activity, affecting transglycosylation with saccha- rides but not alcoholysis. In Paper IV, the mannan oligosaccharide phosphorylase RhMOP130A, first investigated in Paper I was investigated in terms of its structure and studied for its synthetic potential in the reverse phospho- rlysis reaction direction, allowing for elongation of mannan oligosaccharide using activated mannose phosphate. RhMOP130A showed the ability to elongate allyl mannose, such as produced in Paper II, making it an interesting tool for modification of novel glycosides.
The findings of this thesis enhance our comprehension of the molecular factors and reaction parameters that impact the effective conversion of β-mannans into novel glycosides. This showcases the viability of utilizing β- mannanases in the enzymatic synthesis of novel bio-materials.