galactmannan degradation by fungi and gut bacteria: structural enzymology and fine -tuned substrate specifcity

AbstractThe degradation of plant based β-mannan polysaccharides represents one of the many challenges efficiently tackled by microorganisms living in different habitats. In this thesis, glycoside hydrolases (GHs) involved in mannan degradation from two different organisms, Aspergillus nidulans (paper I and II) and Bacteroides ovatus (paper III and IV) were studied. A. nidulans is a saprophytic fungus, while B. ovatus is a symbiotic bacteria present in the human gut. Post-genomic insights into the functionality of the genes in Aspergillus species indicate presence of multiple variants of GHs with same specificity. In paper I and II multiple variants of β-mannosyl hydrolases from GH2 and GH5 were characterised to reveal differences in their fine-tuned substrate specificities. Differences regarding sensitivity to substrate substitutions and length (paper I), general ability of transglycosylation (paper II) was observed. GH2 β-mannosyl hydrolases are β-mannosidases with exo-activity. In paper I differences in fine-tuned substrate specificity among Aspergillus β-mannosidase homologs were studied with parallel bioinformatic and biochemical analysis of representative enzymes. In paper II, three GH5 β-mannanase isozymes AnMan5A, AnMan5B and AnMan5C from A. nidulans were characterised with respect to different acceptor specificities in transglycosylation reactions. Insights into the mannan conversion in human gut were revealed in paper III and IV, involving characterisation of GHs from mannan utilisation locus in B. ovatus. Comparison of product profiles of two GH26 β-mannanases: BoMan26A and BoMan26B from mannan utilisation locus in B. ovatus indicate different product profiles while hydrolysing galactomannans. Periplasmic BoMan26A is more efficient in hydrolysing manno-oligosaccharides and is restricted by galactose substitutions. However, both the β-mannanases produce mannobiose as the main product. Crystal structure of BoMan26A reported in paper III also adds to the structure-function relation of GH26 β-mannanases. A GH36 α-galactosidase, BoGal36A characterised from the same genetic locus efficiently removes galactose substituents from mannan backbone and galactosylated manno-oligosaccharides (paper IV). Schematic representation of galactomannan degradation in B. ovatus is presented in paper III. The rationale for the sequential attack of GHs is based on the substrate preferences, product profiles and cellular location of all the three GHs characterised in paper III and paper IV. Detailed characterisation of the galactomannan degrading enzymes revealing their fine-tuned substrate specificities adds to the knowledge of mannan utilisation which increases the applicability of these enzymes. For instance, in paper II we identify β-mannanases with different transglycosylation specificities. A β-mannanase from this study can be potentially used for synthesis of alkyl glycosides with surfactant properties that can be used in biodegradable detergents. Characterised GH36 α-Galactosidase, BoGal36A could be used for modifying the polysaccharide properties of galactomannans as exemplified in paper IV. (Less)