An alternative broad-specificity pathway for glycan breakdown in bacteria.

The vast majority of glycosidases characterized to date follow one of the variations of the 'Koshland' mechanisms1 to hydrolyse glycosidic bonds through substitution reactions. Here we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities2. Using this, we identify a cluster of enzymes with extremely broad substrate specificities and thoroughly characterize these, mechanistically and structurally. These enzymes not only break glycosidic linkages of both α and ß stereochemistry and multiple connectivities, but also cleave substrates that are not hydrolysed by standard glycosidases. These include thioglycosides, such as the glucosinolates from plants, and pseudoglycosidic bonds of pharmaceuticals such as acarbose. This is achieved through a distinct mechanism of hydrolysis that involves oxidation/reduction and elimination/hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. Homologues of these enzymes occur in both Gram-positive and Gram-negative bacteria associated with the gut microbiome and other body parts, as well as other environments, such as soil and sea. Such alternative step-wise mechanisms appear to constitute largely unrecognized but abundant pathways for glycan degradation as part of the metabolism of carbohydrates in bacteria.

Complex alpha and beta mannan foraging by the human gut bacteria.

The human gut microbiota (HGM), a community of trillions of microbes, underscores its contribution by impacting many facets of host health and disease. In the HGM, Bacteroidota and Bacillota represent dominant bacterial phyla, which mainly rely on the glycans recalcitrant to host digestion to meet their energy requirements. Accordingly, the impact of dietary and host-derived glycans in the assembly and operation of these dominant microbial communities continues to be an area of active research. Among various glycans, mannans represent an integral component of the human diet. Apart from their health effects, the diverse and complex mannan structures bears molecular signatures that alter the expression of specific gene clusters in selected Bacteroidota and Bacillota species. Both the phyla possess variable and sophisticated loci of mannan sensing proteins, hydrolytic enzymes, transporters, and other metabolic proteins to sense, capture and utilize mannans as an energy source. The current review summarizes mannan structural diversity, and strategies opted by select bacterial species of the HGM to forage mannans by focusing primarily on glycoside hydrolases and their effects on host health and metabolism.

Soluble and Cross-Linked Aggregated Forms of α-Galactosidase from Vigna mungo Immobilized on Magnetic Nanocomposites: Improved Stability and Reusability.

α-Galactosidases hold immense potential due to their biotechnological applications in various industrial and functional food sectors. In the present study, soluble and covalently cross-linked aggregated forms of a low molecular weight, thermo-labile α-galactosidase from Vigna mungo (VM-αGal) seeds were immobilized onto chitosan-coated magnetic nanoparticles for improved stability and repeated usage by magnetic separation. Parameters like precipitants (type, amount, and ratio), glutaraldehyde concentration, and enzyme load were optimized for the preparation of chitosan-coated magnetic nanocomposites of cross-linked VM-αGal (VM-αGal-MC) and VM-αGal (VM-αGal-M) resulted in 100% immobilization efficiency. Size and morphology of VM-αGal-M were studied through dynamic light scattering (DLS) and scanning electron microscopy (SEM), while Fourier transform infrared spectroscopy (FTIR) was used to study the chemical composition of VM-αGal-MC and VM-αGal-M. VM-αGal-MC and VM-αGal-M were found more active in a broad range of pH (3-8) and displayed optimal temperatures up to 25 °C higher than VM-αGal. Addition of non-ionic detergents (except Tween-40) improved VM-αGal-MC activity by up to 44% but negatively affected VM-αGal-M activity. Both VM-αGal-MC (15% residual activity after 21 min at 85 °C, Ed 92.42 kcal/mol) and VM-αGal-M (69.0% residual activity after 10 min at 75 °C, Ed 39.87 kcal/mol) showed remarkable thermal stability and repeatedly hydrolyzed the substrate for 10 cycles.

GH36 α-galactosidase from Lactobacillus plantarum WCFS1 synthesize Gal-α-1,6 linked prebiotic α-galactooligosaccharide by transglycosylation.

α-Galactosidases are potent industrial glycoside hydrolases which are relatively less explored for their transglycosylation potential, especially from Lactobacillus genera. A GH36 α-galactosidase from Lactobacillus plantarum WCFS1 was cloned and over expressed in Hi-control Escherichia coli BL21(DE3). Ni-NTA affinity gel chromatography resulted in purified α-galactosidase (LpαG; specific activity 3077.35 U mg-1) having a monomeric weight of ~80 kDa with 29.3% yield. Size exclusion chromatography of LpαG showed native molecular mass of ~240.5 kDa. LpαG displayed optimum activity at pH 6 and 37 °C. The Km,Vmax and kcat/Km of LpαG towards pNPαGal were found to be 0.93 mM and 714.3 µmol ml-1 min-1 and 12,075 s-1 mM-1, respectively. LpαG displayed maximum transglycosylation activity towards melibiose substrate (as both donor and acceptor) and synthesized majorly a trisaccharide with 0.26 mg ml-1 yield. Nuclear magnetic resonance (NMR) characterization revealed that trisaccharide consist of only single species of α-linked galactooligosaccharide (manninotriose; α-d-Galp-(1 â†’ 6)-α-d-Galp-(1 â†’ 6)-d-Glcp) with α-(1 â†’ 6) regioselectivity. Manninotriose displayed prebiotic property by supporting the growth of probiotic L. plantarum WCFS1 and Bifidobacteria adolescentis DSM 20083.

Transcriptional analysis of galactomannooligosaccharides utilization by Lactobacillus plantarum WCFS1.

Plant derived galactomannooligosaccharides (GMOS) are an emerging class of prebiotics, but no information is available on their utilization in lactobacilli at the molecular level. The current study aimed at identifying the genetic loci involved in the transport and catabolism of locust bean gum derived GMOS in Lactobacillus plantarum WCFS1. Substrate depletion study showed that L. plantarum WCFS1 can metabolize only short chain GMOS (degree of polymerization; DP ≤ 3). Global transcriptome microarray profiling of L. plantarum WCFS1 revealed differential expression when GMOS or control sugars (glucose, galactose, and mannose) were used as a sole carbohydrate source. Two genetic loci involved in cellobiose (~3.2 kb) and oligo-sucrose (~7.3 kb) utilization in L. plantarum WCFS1 were highly up-regulated up to 8.3 and up to 6.7-fold, respectively by GMOS utilization. qRT-PCR studies of the selected gene clusters showed correlation with microarray data. Altogether, transcriptome and qRT-PCR studies of L. plantarum WCFS1 suggested that un-substituted mannobiose (DP2) might be metabolized by proteins encoded by the cellobiose operon while, substituted DP2 (galactomannose) and DP3 (galactomannobiose) were most likely transported and catabolized by the oligo-sucrose utilization loci encoded proteins.

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties.

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 µmol ml-1 min-1, and 0.413, 0.0026 s-1 mM-1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in ß-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).

Cross-linked enzyme aggregates (CLEAs) and magnetic nanocomposite grafted CLEAs of GH26 endo-ß-1,4-mannanase: Improved activity, stability and reusability.

A comparative study on immobilization of recombinant endo-ß-1,4-mannanase (ManB-1601), using cross-linked aggregated form (MB-C) and novel chitosan magnetic nanocomposites of MB-C (MB-Mag-C) was carried out. FT-IR and Raman spectroscopy were used to confirm the surface modifications while, scanning electron and atomic force microscopy were performed to demonstrate the surface topology and magnetic nature of MB-C and MB-Mag-C. Among MB-C and MB-Mag-C, the former showed better activity and stability in broad range of pH, thermo-stability and kinetic parameters while, the latter showed higher temperature optima and solvent stability. MB-C and MB-Mag-C when compared with free enzyme showed up to 73.2% higher activity (pH 4-9), up to 95.6% higher stability (pH 3-10, 9h incubation at room temperature), up to 15°C higher optimal temperature, higher stability (up to 83%) in the presence of solvents and up to 1.62-fold higher deactivation energy (Ed). Immobilized enzymes were able to repeatedly hydrolyze locust bean gum till 12 cycles and generated predominantly di-, tri- and tetra- species of ß-manno-oligosaccharides.

Structural Characterization and in Vitro Fermentation of ß-Mannooligosaccharides Produced from Locust Bean Gum by GH-26 endo-ß-1,4-Mannanase (ManB-1601).

Size exclusion chromatography of ß-mannooligosaccharides (ß-MOS) mixtures, obtained from ManB-1601 hydrolysis of locust bean gum, resulted in separation of oligosaccharides with various degrees of polymerization (DP 2, 3, and 5). The oligosaccharides were structurally [ESI-MS, FTIR, XRD, TGA, and NMR (1H and 13C)] and functionally (in vitro fermentation) characterized. DP2 oligosaccharide was composed of two species, (A) mannopyranose ß-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose, while DP3 oligosaccharide showed the presence of only one species, i.e. α-d-galactosyl-ß-d-mannobiose. ManB-1601 was capable of cleaving near the branch points in the substrate, resulting in oligosaccharides with galactose at the terminal position apart from attacking unsubstituted ß-1,4-glycosidic linkages. DP2 and DP3 improved the growth of three out of seven species of Lactobacillus while DP5 resulted in poor growth of all Lactobacillus spp. under in vitro conditions. DP2, DP3, and DP5 were found to inhibit the growth of Escherichia coli, Listeria monocytogenes and Salmonella typhi.

Recombinant endo-mannanase (ManB-1601) production using agro-industrial residues: Development of economical medium and application in oil extraction from copra.

Expression of pRSETA manb-1601 construct in Hi-Control Escherichia coli BL21 (DE3) cells improved recombinant endo-mannanase (ManB-1601) production by 2.73-fold (1821±100U/ml). A low-cost, agro-industrial residue supplemented industrial medium for enhanced and economical production of ManB-1601 was developed in two mutual phases. Phase-I revealed the potential of various pre- (induction time: 5h, induction mode: lactose 0.5mM) and post-induction [peptone supplementation: 0.94%(w/v), glycerol 0.123%(v/v)] parameters for enhanced production of ManB-1601 and resulted in 4.61-fold (8406±400U/ml) and 2.53-fold (3.30g/l) higher ManB-1601 and biomass production, respectively. Under phase-II, economization of phase-I medium was carried out by reducing/replacing costly ingredients with solubilized-defatted flax seed meal (S-DFSM), which resulted in 3.25-fold (5926U/ml) higher ManB-1601 production. Industrial potential of ManB-1601 was shown in oil extraction from copra as enzyme treatment led to cracks, peeling, fracturing and smoothening of copra, which facilitated higher (18.75%) oil yield.