Family 3 CBM improves the biochemical properties, substrate hydrolysis and coconut oil extraction by hemicellulolytic and holocellulolytic chimeras.

To understand the influence of family 3 Carbohydrate Binding Module (hereafter CBM3), single (GH5 cellulase; CelB, CelBΔCBM), bi-chimeric [GH26 endo-mannanase (ManB-1601) and GH11 endo-xylanase (XynB); ManB-XynB [1], ManB-XynB-CBM] and tri-chimeric [ManB-XynB-CelB [1], ManB-XynB-CelBΔCBM] enzyme variants (fused or deleted of CBM) were produced and purified to homogeneity. CBM3 did not alter the pH and temperature optima of bi- and tri-chimeric enzymes but improved the pH and temperature stability of ManB in CBM variants of bi-/tri-chimeric enzymes. Truncation of CBM in CelB shifted the pH optimum and increased the melting temperature (Tm 65 â„ƒ). CBM3 improved both substrate affinity (Km) and catalytic efficiency (kcat/Km) of fused enzymes in tri-chimera and CelB but only Km for bi-chimera. Far-UV CD of CelB and bi- and tri-chimeric enzymes suggested that CBM3 improved the α-helical content and compactness in the native state but did not prevent disintegration of secondary structural contents at acidic pH. Steady-state fluorescence studies suggested that under acidic conditions CBM3 prevented the exposure of hydrophobic patches in bi-chimeric protein but could not avert the opening up of chimeric enzyme structure. Aqueous enzyme assisted treatment of mature coconut kernel using single, bi- and tri-chimeric enzymes led to cracks, peeling and fracturing of the matrix and improved the oil yield by up to 22%.

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.

Insights into ß-manno-oligosaccharide uptake and metabolism in Bifidobacterium adolescentis DSMZ 20083 from whole-genome microarray analysis.

Metabolism of non-digestible dietary glycans directly influences the structure and composition of human gut microbiota and, in turn, the host health. ß-Mannans form an integral component of the modern diet as naturally occurring dietary fibre or additives in processed foods. In the present study, in vitro fermentation and TLC studies were used to determine the ability of adult-associated Bifidobacterium adolescentis DSMZ 20083 to utilise ß-manno-oligosaccharides from guar gum, locust bean gum, konjac root, and copra meal generated using GH26 endo-ß-mannanase (ManB-1601). Further, to gain insights into the underlying molecular mechanism, a whole-genome microarray analysis, RT-qPCR, and molecular docking studies were employed to reconstruct the copra meal ß-manno-oligosaccharides (CM-ß-MOS) utilisation pathway in B. adolescentis DSMZ 20083. B. adolescentis DSMZ 20083 grew appreciably (O.D600 nm up to 0.8) on all tested ß-manno-oligosaccharides but maximally on CM-ß-MOS. CM-ß-MOS having DP2-3 were found to deplete from the fermentation media. Whole-genome transcriptome analysis, RT-qPCR, and molecular docking studies suggested that in B. adolescentis DSMZ 20083, ABC & MFS transporters are possibly involved in the uptake of DP ≥ 2 and DP ≥ 3 linear CM-ß-MOS, respectively, while GH1 ß-glucosidase, and GH32 ß-fructofuranosidase possibly cleave linear CM-ß-MOS into monosaccharides. Sugar absorption and utilisation pathways; Bifid shunt, ABC transport system, pyruvate metabolism, glycolysis/gluconeogenesis, pentose, and glucouronate inter-conversions were also found up-regulated following the growth on CM-ß-MOS. This is the first study reporting on possible molecular determinants used by B. adolescentis DSMZ 20083 to utilise ß-manno-oligosaccharides. Our studies can prove resourceful to food and nutraceutical industries, aiming at precision microbiome modulation using ß-manno-oligosaccharides.

Co-culture fermentations suggest cross-feeding among Bacteroides ovatus DSMZ 1896, Lactiplantibacillus plantarum WCFS1 and Bifidobacterium adolescentis DSMZ 20083 for utilizing dietary galactomannans.

Galactomannans from sources like guar, fenugreek, locust bean, and copra form an important part of human diet. In this study, we have attempted to understand the cross-feeding and resource sharing between a generalist degrader and probiotic utilizers for utilizing dietary galactomannans. In mono-cultures, Bacteroides ovatus DSMZ 1896 grew maximally on substituted galactomannans and produced high amount of succinate. Polysaccharide break down products [ß-manno-oligosaccharides; degree of polymerization (DP) 2-4] left after the growth of B. ovatus DSMZ 1896 in galactomannan supplemented media supported the growth of Lactiplantibacillus plantarum WCFS1 (DP2 and DP3) and Bifidobacterium adolescentis DSMZ 20083 (majorly DP3) and led to the production of lactate and acetate, respectively as the major end products. Co-cultures (bi- and tri-cultures) studies demonstrated cross-feeding being used as a strategy for resource sharing among B. ovatus DSMZ 1896, L. plantarum WCFS1 and B. adolescentis DSMZ 20083 while foraging galactomannans. Structure and DP of galactomannan substrates altered the SCFA and organic acid production patterns in co-cultures.

Short-chain ß-manno-oligosaccharides from copra meal: structural characterization, prebiotic potential and anti-glycation activity.

Size-exclusion chromatography, HR-ESI-MS and FT-IR of copra meal hydrolyzed by ManB-1601 showed the presence of oligosaccharides (CM-ß-MOS) having a degree of polymerisation (DP) between 2 and 4. Thermal decomposition studies of the purified CM-ß-MOS (DP 2, 3 and 4) showed mass loss at high temperatures (135.8 °C to 600 °C). DP2, DP3 and DP4 CM-ß-MOS were adjudged as un-substituted Manß-4Man, Manß-4Manß-4Man and Manß-4Manß-4Manß-4Man, respectively, using NMR (1H and 13C) studies. During fermentation, purified CM-ß-MOS supported the growth of Lactobacillus sp. and inhibited enteropathogens (Escherichia coli, Listeria monocytogenes and Salmonella typhi). Acetate was the predominant short-chain fatty acid produced by Lactobacillus sp. RT-PCR studies of L. plantarum WCFS1 fed with CM-ß-MOS showed up-regulation (up to 6.7-fold) of the cellobiose utilization operon (pts23C and pbg6) and oligo-sucrose utilization loci (pts1BCA and agl2). Biochemical (free amino groups, carbonyl and fructosamine content), fluorescence (AGEs-specific and intrinsic) and molecular docking studies suggested the anti-glycation potential of CM-ß-MOS.

Truncation of C-terminal amino acids of GH26 endo-mannanase (ManB-1601) affects biochemical properties and stability against anionic surfactants.

Hitherto, the contribution of C-terminal amino acids in structure, stability and function of GH26 endo-mannanases has not been demonstrated. Semi-logarithmic plot of endo-mannanase activity showed a progressive decline with increase in the number of truncated amino acids [ManB-CΔ5 (129 U/mL), ManB-CΔ10 (47 U/mL), ManB-CΔ15 (0.05 U/mL) and ManB-CΔ20 (0.02 U/mL)]. ManB-CΔ5 and ManB-CΔ10 exhibited similar temperature and pH optima and product profile but biochemical properties (kinetic constants, mannan hydrolysis, response to metal ions and enzyme inhibitors) and stability (in presence of commercial detergents, anionic surfactants and organic solvents and half-life) were markedly affected. Interaction of truncated proteins with anionic surfactants was probed using intrinsic, Nile red, acrylamide quenching, resonance light scattering and synchronous fluorescence spectroscopy studies. Truncation of ten amino acids increased vulnerability to anionic surfactants as conformational changes, exposure of the hydrophobic core and susceptibility to unfolding process were observed. The microenvironment around Trp residues was affected more with surfactants as compared to Tyr residues in truncated proteins. Zn2+ coordination might not play a role in providing stability against SDS. MD simulation studies corroborated that C-terminal amino acids (327-336) helps in structure stabilization, regulating flexibility of loops around the active site and preventing denaturation in the presence of SDS.

Alkali-stable GH11 endo-ß-1,4 xylanase (XynB) from Bacillus subtilis strain CAM 21: application in hydrolysis of agro-industrial wastes, fruit/vegetable peels and weeds.

GH11 endo-xylanases, due to their inherent structural and biochemical properties, are the key to efficient bioconversion of lignocellulosic biomass into value-added products. A GH11 endo-xylanase (XynB) from Bacillus subtilis strain CAM 21 was cloned, over-expressed and purified (Mw∼24 kDa) using Ni-NTA affinity chromatography. XynB showed optimum activity at pH 7.0 and 50°C and was stable (>88%) in a broad range of pH (4-11). The apparent Km, Kcat and Kcat/Km of XynB were 2.9 mg/ml, 1961.2/sec, and 675.62 ml/mg/sec, respectively using birchwood xylan as substrate. XynB was a classical endo-xylanase as it hydrolyzed birchwood xylan to xylo-oligosaccharides and not xylose. Kinetic stability of XynB at 45-53°C was between 43-182 min. Secondary structure analysis of XynB using far-UV CD spectroscopy revealed presence of 51.85% ß strands and 2.64% α helix and was consistent with the homology modeling studies. XynB hydrolyzed the xylan extracted from agro-industrial wastes and fruit/vegetable peels by releasing up to 670 mg/g of reducing sugars. The xylan extracted from weeds (Ageratum conyzoides, Achyranthes aspera and Tridax procumbens) had characteristic signatures of hemicelluloses and after XynB hydrolysis showed cracks, peeling and release of up to 135.2 mg/g reducing sugars.

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.

Molecular advancements on over-expression, stability and catalytic aspects of endo-ß-mannanases.

The hydrolysis of mannans by endo-ß-mannanases continues to gather significance as exemplified by its commercial applications in food, feed, and a rekindled interest in biorefineries. The present review provides a comprehensive account of fundamental research and fascinating insights in the field of endo-ß-mannanase engineering in order to improve over-expression and to decipher molecular determinants governing activity-stability during harsh conditions, substrate recognition, polysaccharide specificity, endo/exo mode of action and multi-functional activities in the modular polypeptide. In-depth analysis of the available literature has also been made on rational and directed evolution approaches, which have translated native endo-ß-mannanases into superior biocatalysts for satisfying industrial requirements.

Zn2+ stapling of N and C-terminal maintains stability and substrate affinity in GH26 endo-mannanase.

Metal binding sites are present in one-third of proteins and are crucial for biological functions and structural maintenance. GH26 endo-mannanase (ManB-1601) from Bacillus sp. harbors a Zn2+ binding site which connects N (H1, H23) and C (E336)-terminal residues. Present study reveals how native circularization of ManB-1601 through Zn2+ coordination regulates the structure-function. We generated individual Zn2+ coordinating mutants and characterized them using biochemical and biophysical approaches. Contribution of individual Zn2+ coordination towards maintaining ManB-1601 stability and rigidity was in the following order H23>H1 > E336. Elimination of E336 and H23-Zn2+ coordination affected substrate hydrolysis to a greater degree than H1-Zn2+ coordination. Metal quantification of mutant proteins indicated that H23A did not contain Zn2+. Molecular dynamic simulation studies revealed disruption of H23-Zn2+ coordination leads to increased flexibility of N and C-terminal, active site loops and consequent drifting of substrate away from the active site region. Finally, mechanistic understanding on the functioning of Zn2+ site in ManB-1601 is developed wherein 1) H23 by anchoring Zn2+ ion majorly regulates the structure-function properties, 2) H1 provides thermo-stability, 3) E336 contributes towards maintaining substrate hydrolysis.

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.

Structural diversity and prebiotic potential of short chain ß-manno-oligosaccharides generated from guar gum by endo-ß-mannanase (ManB-1601).

Size exclusion chromatography of short chain ß-manno-oligosaccharides (GG-ß-MOS) produced after endo-mannanase (ManB-1601) hydrolysis of guar gum resulted in seven (P1-P7) peaks. Electron spray ionization mass-spectrometry (ESI-MS) revealed P3, P4, P5 and P6 peaks as pentasaccharide (DP5), tetrasaccharide (DP4), trisaccharide (DP3) and disaccharide (DP2), respectively. DP2 and DP3 GG-ß-MOS were structurally characterized by NMR (1H and 13C), FTIR and XRD. DP2 GG-ß-MOS was composed of two species (A) mannopyranose ß-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose while, DP3 oligosaccharide showed presence of three species i.e. (A) α-d-galactosyl-ß-d-mannobiose (galactosyl residue at reducing end), (B) α-d-galactosyl-ß-d-mannobiose (galactosyl residue at non-reducing end) and (C) mannopyranose ß-1,4 mannose ß-1,4 mannopyranose. In batch fermentation, DP2 GG-ß-MOS was preferred over DP3 by all Lactobacillus sp. except Lactobacillus casei var rhamnosus. DP2/DP3 and GG-ß-MOS mixture inhibited the growth of enteropathogens in monoculture and co-culture fermentations, respectively. Fermentation of GG-ß-MOS mixture by Lactobacillus sp. produced short chain fatty acids.

Salt bridges are pivotal for the kinetic stability of GH26 endo-mannanase (ManB-1601).

Hitherto, how salt bridges contribute towards the structure-function of endo-mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo-mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endo-mannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and E171/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (∆Tm 0 to -5 °C) and conformational stability (∆∆G -0.37 to -1.25 Kcal mol-1) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T1/2) at 66 °C (1.1 to 6-fold) and 70 °C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural elements for enzyme functioning by contributing towards kinetic stability.

How substrate subsites in GH26 endo-mannanase contribute towards mannan binding.

Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that -3, -2, -1, +1 and + 2 subsites have a major role while, -4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of -1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that -1 and -2 subsite mutants had 27- and 30-fold higher binding affinity (KD) for carob galactomannan when compared with catalytic mutant. The -1 subsite mutant also had highest KD values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher KD) and ivory nut mannan (up to 4.3-fold higher KD) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, -3 mutant displayed 10-fold higher KD for both carob galactomannan and glucomannan while, -4 mutant did not show any noticeable change in KD values when compared to catalytic mutant.