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

Conversion of aldoses to valuable ω-amino alcohols using amine transaminase biocatalysts

The conversion of readily available monosaccharides to high-value amino alcohols using a key biocatalytic step is an attractive strategy for the preparation of these chiral synthons. Here, we report a previously undescribed example of the direct amination of monosaccharides, which exist predominantly in their cyclic form at equilibrium, using amine transaminase biocatalysts, providing access to a panel of amino alcohols in moderate to high conversion and isolated yield. A recently developed high-throughput colorimetric screen, employing o-xylylenediamine, was initially used to identify amine transaminase enzymes displaying this activity towards cyclic sugars and reactions were successfully scaled-up using isopropylamine

Application of 'smart' amine donors for rapid screening and scale-up of transaminase-mediated biotransformations

The 'smart' amine donors o?xylylenediamine and cadaverine were employed for the rapid screening of a large ketone library and subsequent preparative?scale synthesis of selected compounds using the commercially available amine transaminase, ATA256. The methodology enables both screening and preparative?scale biotransformations to be performed with a single enzyme and simplifies the generation of sp3?rich small molecule libraries

Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

AbstractThe thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content

The GH5 1,4-β-mannanase from <i>Bifidobacterium animalis</i> subsp. <i>lactis </i>Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8)

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

The gene encoding beta-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 mu M(8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, kappa(cat) was determined to be 444 s(-1) and K-m 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides

Development of a Corynebacterium glutamicum bio-factory for self-sufficient transaminase reactions

Grigoriou S, Kugler P, Kulcinskaja E, et al. Development of a Corynebacterium glutamicum bio-factory for self-sufficient transaminase reactions. Green Chemistry. 2020;22:4128-4132

The physico-chemical properties of dietary fibre determine metabolic responses, short-chain Fatty Acid profiles and gut microbiota composition in rats fed low- and high-fat diets.

The aim of this study was to investigate how physico-chemical properties of two dietary fibres, guar gum and pectin, affected weight gain, adiposity, lipid metabolism, short-chain fatty acid (SCFA) profiles and the gut microbiota in male Wistar rats fed either low- or high-fat diets for three weeks. Both pectin and guar gum reduced weight gain, adiposity, liver fat and blood glucose levels in rats fed a high-fat diet. Methoxylation degree of pectin (low, LM and high (HM)) and viscosity of guar gum (low, medium or high) resulted in different effects in the rats, where total blood and caecal amounts of SCFA were increased with guar gum (all viscosities) and with high methoxylated (HM) pectin. However, only guar gum with medium and high viscosity increased the levels of butyric acid in caecum and blood. Both pectin and guar gum reduced cholesterol, liver steatosis and blood glucose levels, but to varying extent depending on the degree of methoxylation and viscosity of the fibres. The medium viscosity guar gum was the most effective preparation for prevention of diet-induced hyperlipidaemia and liver steatosis. Caecal abundance of Akkermansia was increased with high-fat feeding and with HM pectin and guar gum of all viscosities tested. Moreover, guar gum had distinct bifidogenic effects independent of viscosity, increasing the caecal abundance of Bifidobacterium ten-fold. In conclusion, by tailoring the viscosity and possibly also the degree of methoxylation of dietary fibre, metabolic effects may be optimized, through a targeted modulation of the gut microbiota and its metabolites

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08 juin 19421942/06/08 (A72,N207).Appartient à l’ensemble documentaire : PoitouCh