Galactomannan degradation by fungi and gut bacteria

Projekt: Avhandling

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The 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 and Bacteroides ovatus 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. 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 , general ability of transglycosylation 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. 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 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. Schematic representation of galactomannan degradation in B. ovatus is proposed. 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.
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, 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 the study

Populärvetenskaplig beskrivning

Complex carbohydrate structures are one of the major components of plant cell walls. They represent major renewable organic matter in nature and can potentially be used for production of various valuable products in a sustainable way, for e.g. biofuels, chemicals, bio-degradable detergents etc. However, cost effective utilisation of this renewable organic matter still remains as a challenge. Interestingly, microorganisms from different environmental niches like soil and human gut utilise these complex carbohydrates in an effective way for various purposes. They express an array of enzymes, which acts cooperatively to modify or degrade the complex polysaccharides. Some of these enzymes are specific to a substrate, and are fine-tuned to tackle the complexity of the carbohydrate structure. Identification of these differences in fine-tuning and optimisation of enzyme mixtures have the potential to increase the applicability of enzyme conversion.
β-Mannans are plant based hemicellulosic polysaccharides with a mannose backbone. Mannans are present in various forms, for example linear mannans and substituted mannans, such as glucomannans and galactoglucomannan. Apart from being used as food thickeners and additives in food industries, mannans also have promising potential for other applications like packaging, drug delivery agents and prebiotics etc. Enzymes involved in modification of β-mannans are biotechnologically important in two perspectives: for hydrolysis of hemicellulosic mannans in the pre-treatment step of energy production and for synthesis or modification of mannan polymers for other applications. These enzymatic modifications of mannans require enzymes with well characterised properties. The focus of this thesis is to study the mannan degrading enzymes from microorganisms present in different natural habitats, i.e. the saprophytic habitat and human gut. These enzymes can serve as tools for above mentioned applications. Initial selection involved screening of microorganisms that can effectively utilise galactomannan as the carbon source based on the available data like genomics, growth and protein expression.
Aspergillus species are considered as biotechnologically important organisms and are known for their hemicellulose degradation potential. Many enzymes from Aspergillus species are also commercially available for biotechnological applications. Post-genomic insights into the functionality of the genes in Aspergillus species indicate presence of multiple variants of enzymes with the same basic activity. These multiple variants of enzymes may look redundant at first sight, but given the complexity of the mannan polysaccharides, they may have subtle differences in their activity. Therefore, in this study (paper I and II) multiple variants of enzymes with the same basic activity were extensively characterised to reveal differences in their fine–tuned substrate specificity, based on their natural substrate preferences. Such detailed characterisation of the galactomannan degrading enzymes adds to the knowledge of the mannan utilisation toolkit, which increases the applicability of these enzymes. For instance, in paper II we identify β-mannanases that can potentially be used for synthesis of bio-degradable surfactants in detergents. A novel α-galactosidase characterised in paper IV can be used to modify galactomannan properties needed during various biotechnological applications.
Polysaccharide conversion in the human gut is one of the hot topics addressed by many scientists in the world. Very limited information is available related to mannan conversion in the human gut. Given that mannans are present in food, further on in this study enzymes involved in the mannan conversion in the human gut were characterised. Bacteroides are one of the dominant bacteria in human gut. They are proposed to have discrete genetic loci specific for utilising specific polysaccharide. Recently, it was shown that Bacteroides ovatus, a human gut symbiont, can grow on galactomannan as carbon source and also has a discrete genetic locus specific to mannan polysaccharide utilisation. Characterisation of the enzymes involved in galactomannan degradation from this locus (paper III and IV), along with determining their cellular location, helps in understanding the mannan degradation in human gut. This may further help in planning mannan based prebiotics, therapeutics, drug delivery agents etc. Additionally, the analysed properties and functionalities of the enzymes described in this thesis will also help in obtaining optimised enzyme cocktails for complete degradation of diverse mannan structures by cooperative action between the different mannan degrading enzymes.
Kort titelstructural enzymology and fine -tuned substrate specificity
StatusSlutfört
Gällande start-/slutdatum2011/01/012016/06/10

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