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Mannotriose O-MTR
Product code: O-MTR

50 mg

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Content: 50 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 28173-52-6
Molecular Formula: C18H32O16
Molecular Weight: 504.4
Purity: > 95%
Substrate For (Enzyme): endo-1,4-β-Mannanase

High purity Mannotriose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

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Megazyme publication

Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.

Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

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Production of mannooligosaccharides producing β-Mannanase by newly isolated Penicillium aculeatum APS1 using oil seed residues under solid state fermentation.

Bangoria, P., Divecha, J. & Shah, A. R. (2021). Biocatalysis and Agricultural Biotechnology, 34, 102023.

The present investigation was focused on the production of extracellular β-mannanase from newly isolated Penicillium aculeatum APS1 using palm kernel cake and soyabean meal which are the residual by-products of oil extraction industry, under solid state fermentation. On supplementing palm kernel cake with 20% soyabean meal, yield of β-mannanase production was reached to 2807 U/g. Response surface methodology was used for statistical optimization of β-mannanase production. Two independent variables, namely moisture level and incubation time, were found to be significantly contributing for the production process. Under optimized condition of moisture level (52.25%) and incubation time (130 h), the yield of β-mannanase was improved by 1.6 fold and maximum activity of β-mannanase was 4696 U/g. The optimum temperature and pH for crude β-mannanase were 65°C and 6.0, respectively. Crude and partially purified β-mannanase was found to be effective in release of mannooligosachharides by hydrolysis of mannan rich substrates viz. locust bean gum, guar gum and konjac glucomannan. Qualitative and quantitative analysis of MOS was carried out by TLC and ion chromatography. β-Mannanase from Penicillium aculeatum APS1 was found to have properties suitable for applications in feed/food industry.

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High efficient degradation of glucan/glucomannan to cello-/mannan-oligosaccharide by endoglucanase via tetrasaccharide as intermediate.

Miao, T., Basit, A., Wen, J., Liu, J., Zheng, F., Cao, Y. & Jiang, W. (2021). Food Chemistry, 129175.

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.

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Production of manno-oligosaccharide from Gleditsia microphylla galactomannan using acetic acid and ferrous chloride.

Xu, W., Han, M., Zhang, W., Zhang, F., Lei, F., Wang, K. & Jiang, J. (2021). Food Chemistry, 346, 128844.

A novel and efficient method for manno-oligosaccharides (MOS) production has been proposed by utilizing Gleditsia microphylla galactomannan as the starting material. This co-operative hydrolysis using ferrous chloride (Fe2+) and acetic acid (HAc) effectively improved the MOS yield and meanwhile decreased the amount of monosaccharide and the 5-hydroxymethyl-furfural (HMF). The highest yields under the optimum conditions were 46.7% by HAc hydrolysis (5 M HAc at 130°C for 120 min); 37.3% by Fe2+ hydrolysis (0.1 M Fe2+ at 150°C for 120 min); and 51.4% by co-operative hydrolysis (2 M HAc, 0.05 M Fe2+ at 160°C for 10 min). From the changes in the value of M/G (mannose/galactose) ratios, it was deduced that Fe2+ predominantly cleaves the main chain, and HAc assists in the breakage of the side chain, thus resulting in the high-efficient co-operative hydrolysis for the production of MOS.

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Production of mannooligosaccharides from various mannans and evaluation of their prebiotic potential.

Suryawanshi, R. K. & Kango, N. (2021). Food Chemistry, 334, 127428.

Aspergillus quadrilineatus endo-β-mannanase effectively degraded konjac glucomannan (66.09% w/v), copra meal (38.99% w/v) and locust bean galactomannan (20.94% w/v). High performance liquid chromatography (HPLC) analysis of KG hydrolysate indicated its mannooligosaccharides (MOS) content (656.38 mg/g) with high amounts of DP 5 oligosaccharide. Multi-scale characterization of mannan hydrolysate was done using FTIR and 13C NMR which revealed α and β form of galactose or glucose in MOS, respectively. CM and LBG hydrolysates (1 mg/mL) have shown cytotoxic effect and reduced cell viability of Caco-2 cells by 45% and 62%, respectively. MOS DP (1–4) derived from LBG supported better Lactobacilli biofilm formation as compared to KG hydrolysate containing high DP MOS (5–7). Lactobacilli effectively fermented MOS to generate acetate and propionate as main short chain fatty acids. Lactobacilli produced leucine, isoleucine and valine as branched chain amino acids when grown on LBG hydrolysate.

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Engineering of β-mannanase from Aspergillus niger to increase product selectivity towards medium chain length mannooligosaccharides.

Arunrattanamook, N., Wansuksri, R., Uengwetwanit, T. & Champreda, V. (2020). Journal of Bioscience and Bioengineering, In Press.

Mannooligosaccharides (MOSs) are one of the most commonly used biomass-derived feed additives. The effectiveness of MOS varies with the length of oligosaccharides, medium length MOSs such as mannotetraose and mannopentaose being the most efficient. This study aims at improving specificity of β-mannanase from Aspergillus niger toward the desirable product size through rational-based enzyme engineering. Tyr 42 and Tyr 132 were mutated to Gly to extend the substrate binding site, allowing higher molecular weight MOS to non-catalytically bind to the enzyme. Hydrolysis product content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. Instead of mannobiose, the enzyme variants yielded mannotriose and mannotetraose as the major products, followed by mannobiose and mannopentaose. Overall, 42% improvement in production yield of highly active mannotetraose and mannopentaose was achieved. This validates the use of engineered β-mannanase to selectively produce larger MOS, making them promising candidates for large-scale MOS enzymatic production process.

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High-efficiency expression of a superior β-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides.

Liu, Z., Ning, C., Yuan, M., Fu, X., Yang, S., Wei, X., Xiao, M., Mou, H. & Zhu, C. (2020).  Bioresource Technology, 123482.

β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg−1) and MEIR (9003.1 U mg−1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg−1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL−1). The prebiotic mannooligosaccharides (< 2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.

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Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function.

Li, A., Laville, E., Tarquis, L., Lombard, V., Ropartz, D., Terrapon, N., Henrissat, B., Guieysse, D., Esque, J., Durand, J., Diego P. & Potocki-Veronese, D. P. (2020). Microbial Genomics, mgen000404.

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

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The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production.

Rahmani, N., Amanah, S., Santoso, P. & Lisdiyanti, P. (2020). Biocatalysis and Agricultural Biotechnology, 24, 101532.

Actinomycetes have been identified as one of the most diverse groups of microorganisms that play a vital role in the production of enzymes and nutraceuticals. In addition, prior studies on the wild type strain of Kitasatospora sp. emphasized on its ability to exhibit high β-mannanase activity. This study aimed to purify, characterize and evaluate the potential of this strain in the production of mannooligosaccharides using mannan polymer. The enzyme was produced by submerged fermentation of a medium contains locus bean gum as a carbon source. The crude mannanase was subjected to polyethylene glycol precipitation and ion exchange chromatography. The completion of the purification process was confirmed by SDS-PAGE and purified of enzyme characterization were investigated, analysis hydrolysis product was conducted by TLC. The enzymes exhibited the activity of 37.0 U/mL. A purification factor of 1.4-fold was achieved with specific activity of 6.3 U/mg. An increase of activity was recorded from 15.0 U/mL and 4.4 U/mL to 19.3 U/mL and 6.3 U/mL. In addition, the total protein decreased from 338.5 mg/mL to 45.7 mg/mL. The purified β-mannanase has the molecular weight was approximately 37.0 kDa with optimal activity at pH 6.0 and 60°C and relatively stability at a pH variety of 6.0-9.0, retaining > 90% activity. This product was capable of hydrolysing various mannan polymers (porang potato, palm sugar fruit, coconut cake, palm cernel cake) and other commercial mannan (LBG, β-mannan, konjac, ivory nut), subsequently producing various sizes of mannoligosaccharides and mannose potential for food and feed industry.

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Characteristics and bioactive properties of mannooligosaccharides derived from agro-waste mannans.

Jana, U. K. & Kango, N. (2020). International Journal of Biological Macromolecules, 149, 931-940.

Mannooligosaccharides (MOS) were derived using Aspergillus oryzae β-mannanase (ManAo) from different mannan-rich agro-wastes, palm kernel cake (PKC), guar gum and copra meal (CM). Guar gum (GG) released higher amount of MOS (56.31% w/w) from which purification of mannobiose (0.68 mg) and mannotriose (1.26 mg) was demonstrated using size-exclusion chromatography. FTIR analysis of mannan hydrolysates showed characteristic peaks in 1200–900 cm−1 region indicating the presence of MOS. 1H & 13C NMR spectra showed presence of anomeric sugar forms of MOS in different mannan hydrolysates. MOS from locust bean gum and guar gum had both α- and β-anomers while PKC and CM had only α-anomer. Growth promotional activities of different MOS were demonstrated using two probiotic Lactobacilli. Besides, enzymatically derived MOS also showed metal (Fe2+) chelating and anti-oxidant activities, wherein best anti-glycating agent was evaluated as MOS from PKC. PKC derived MOS showed highest cytotoxicity (74.19%) against human colon adenocarcinoma cell line (Caco-2). This study demonstrated the prebiotic potential of agro-waste derived MOS and possibility of their utilization as a functional food ingredient.

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Copra meal hydrolysis by the recombinant β-mannanase KMAN-3 and MAN 6.7 expressed in Escherichia coli.

Sritrakul, N., Nitisinprasert, S. & Keawsompong, S. (2020). 3 Biotech, 10(2), 44.

Hydrolysis products of defatted copra meal (DCM) hydrolysis were investigated with either recombinant β-mannanases from Klebsiella oxytoca KUB-CW2-3 (KMAN-3) or Bacillus circulans NT 6.7 (MAN 6.7). Morphological changes and functional groups of solid residues were also determined by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Results revealed that the Michaelis–Menten constant (Km) and maximum velocity (Vmax) values of KMAN-3 on DCM were 2.4 mg/ml and 5.4 U/mg, respectively, while MAN 6.7 recorded Km and Vmax at 2.0 mg/ml and 4.3 U/mg, respectively. Both enzymes efficiently randomly hydrolysed DCM and produced a range of different manno-oligosaccharides (MOS). The profile of hydrolysis products was different for each enzyme used. Main products from hydrolysis of DCM by KMAN-3 and MAN 6.7 were various MOS including mannobiose (M2), mannotriose (M3), mannotetraose (M4), and mannose, whereas mannopentaose (M5) was only found from KMAN-3. Amount of M3 produced by KMAN-3 was about three times higher than from MAN 6.7. Total MOS yield for KMAN-3 was 1.5-folds higher than for MAN 6.7. SEM analysis showed that enzymatic hydrolysis with KMAN-3 and MAN 6.7 resulted in deconstruction of the DCM structure which generated a variety of MOS products. FTIR spectra revealed that the properties of both hydrolysed solids were not significantly different compared to the original DCM. Results suggested that KMAN-3 was a promising candidate for production of high MOS content from copra meal.

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Preparation, characterization, and prebiotic activity of manno-oligosaccharides produced from cassia gum by a glycoside hydrolase family 134 β-mannanase.

Li, Y. X., Liu, H. J., Shi, Y. Q., Yan, Q. J., You, X. & Jiang, Z. Q. (2020). Food Chemistry, 309, 125709.

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 β-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL-1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50°C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1-F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α-d-galactosyl-β-D-mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2-3 rather than those with DP 4-5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.

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High-level expression of a thermophilic and acidophilic β-mannanase from Aspergillus kawachii IFO 4308 with significant potential in mannooligosaccharide preparation.

Liu, Z., Ning, C., Yuan, M., Yang, S., Wei, X., Xiao, M., Fu, X., Zhu, C. & Mou, H. (2020). Bioresource Technology, 295, 122257.

An engineered thermophilic and acidophilic β-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme β-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80°C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (<2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.

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Economic production of thermo-active endo β-mannanase for the removal of food stain and production of antioxidant manno-oligosaccharides.

Ismail, S. A., Hassan, A. A. & Emran, M. A. (2019). Biocatalysis and Agricultural Biotechnology, 22, 101387.

β-Mannanases are β-1,4-mannan-glycosidic bonds hydrolyzing enzymes that participate in various biotechnological applications. In the current study, the production of the enzyme was performed by solid state fermentation of rice straw using the locally isolated fungus Trichoderma longibrachiatum RS1 and the production of the enzyme was optimized to reach 89.73U/g dry substrate. The isolated fungus was identified on the base of its cultural and morphological features and by 18S rDNA sequencing. The optimum temperature for the activity of the partially purified enzyme was indicated to be 75°C. Although production of fungal β-mannanases have been previously studied but the production of thermo-active enzymes are still challengeable. The Vmax and Km were 6.2U/mg protein/min and 3.33 mg/mL respectively, indicating the comparatively high affinity of the produced enzyme toward mannan substrates. The thermal stability of the produced enzyme estimated that its half lives were 633.01, 50.77 and 20.25 min−1 at 55, 60 and 65°C respectively. The produced enzyme can be efficiently used in the removal of mannan based food stain. Moreover, the efficiency of the produced enzyme in the production of manno-oligosaccharides by the hydrolysis of mannan polymers was examined. The results indicated the release of 1.8 and 0.66 mg reducing sugar/mL by the hydrolysis of locust bean and guar gum for 2 h with hydrolysis percentage of 27 and 9.9% respectively. Finally, the produced manno-oligosaccharides were examined for their antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl free radical.

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Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

Álvarez-Cao, M. E., Cerdán, M. E., González-Siso, M. I., & Becerra, M. (2019). Microbial Cell Factories, 18(1), 1-17.

Background: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

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

Mary, P. R., Prashanth, K. H., Vasu, P. & Kapoor, M. (2019). Carbohydrate Research, 486, 107822.

Size exclusion chromatography of short chain β-manno-oligosaccharides (GG-β-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-β-MOS were structurally characterized by NMR (1H and 13C), FTIR and XRD. DP2 GG-β-MOS was composed of two species (A) mannopyranose β-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose while, DP3 oligosaccharide showed presence of three species i.e. (A) α-d-galactosyl-β-d-mannobiose (galactosyl residue at reducing end), (B) α-d-galactosyl-β-d-mannobiose (galactosyl residue at non-reducing end) and (C) mannopyranose β-1,4 mannose β-1,4 mannopyranose. In batch fermentation, DP2 GG-β-MOS was preferred over DP3 by all Lactobacillus sp. except Lactobacillus casei var rhamnosus. DP2/DP3 and GG-β-MOS mixture inhibited the growth of enteropathogens in monoculture and co-culture fermentations, respectively. Fermentation of GG-β-MOS mixture by Lactobacillus sp. produced short chain fatty acids.

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Ethanol Precipitation of Mannooligosaccharides from Subcritical Water-Treated Coconut Meal Hydrolysate.

Klinchongkon, K., Bunyakiat, T., Khuwijitjaru, P. & Adachi, S. (2019). Food and Bioprocess Technology, 12(7), 1197-1204.

Subcritical water hydrolysis is an effective method for producing mannooligosaccharides from coconut meal, which is a by-product from coconut milk processing. In this study, the purification process to obtain mannooligosaccharides from coconut meal hydrolysate was investigated. The effects of adsorbent (activated carbon and bentonite), concentration (1-10% w/v), and adsorption time (5-60 min) were studied for impurities removal. The activated carbon showed much higher efficiency for impurities removal. Mannooligosaccharides were precipitated using ethanol at different concentrations (0-90% v/v) and initial carbohydrate contents (50, 100, and 200 g/L). The results showed that the ethanol concentration at 90% v/v and initial carbohydrate content of 200 g/L gave the highest recovery of saccharides (31 g/L). The obtained precipitate contained 9.7, 22.6, 12.9, 19.4, 19.4, and 16.1% w/w of saccharides with 1 to 6 degree of polymerization, respectively.

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Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on β‐mannan possess distinct manno‐oligosaccharide‐binding profiles.

Ejby, M., Guskov, A., Pichler, M. J., Zanten, G. C., Schoof, E., Saburi, W., Slotboom, D. J. & Abou Hachem, M. (2019). Molecular Microbiology, 112(1), 114-130.

Human gut bifidobacteria rely on ATP‐binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide‐specific solute‐binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the β‐manno‐oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (Kd≈80 nM), affinity of BlMnBP1 is up to 2570‐fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1‐like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan‐degrading Bacteroides ovatus strain in co‐cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC‐transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic.

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Constitutive expression and cell-surface display of a bacterial β-mannanase in Lactobacillus plantarum.

Nguyen, H. M., Pham, M. L., Stelzer, E. M., Plattner, E., Grabherr, R., Mathiesen, G., Peterbauer, C. K., Haltrich, D. & Nguyen, T. H. (2019). Microbial Cell Factories, 18(1), 76.

Background: Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production. Results: Two strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS). Conclusion: This work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

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Enzymatic synthesis and polymerisation of β-mannosyl acrylates produced from renewable hemicellulosic glycans.

Rosengren, A., Butler, S. J., Arcos-Hernandez, M., Bergquist, K. E., Jannasch, P. & Stålbrand, H. (2019). Green Chemistry, 21(8), 2104-2118.

We show that glycoside hydrolases can catalyse the synthesis of glycosyl acrylate monomers using renewable hemicellulose as a glycosyl donor, and we also demonstrate the preparation of novel glycopolymers by radical polymerisation of these monomers. For this, two family 5 β-mannanases (TrMan5A from Trichoderma reesei and AnMan5B from Aspergillus niger) were evaluated for their transglycosylation capacity using 2-hydroxyethyl methacrylate (HEMA) as a glycosyl acceptor. Both enzymes catalysed conjugation between manno-oligosaccharides and HEMA, as analysed using MALDI-ToF mass spectrometry (MS) as an initial product screening method. The two enzymes gave different product profiles (glycosyl donor length) with HEMA, and with allyl alcohol as acceptor molecules. AnMan5A appeared to prefer saccharide acceptors with lower intensity MS peaks detected for the desired allyl and HEMA conjugates. In contrast to AnMan5A, TrMan5A showed pronounced MS peaks for HEMA-saccharide conjugation products. TrMan5A was shown to catalyse the synthesis of β-mannosyl acrylates using locust bean gum galactomannan or softwood hemicellulose (acetyl-galactoglucomannan) as a donor substrate. Evaluation of reaction conditions using galactomannan as a donor, HEMA as an acceptor and TrMan5A as an enzyme catalyst was followed by the enzymatic production and preparative liquid chromatography purification of 2-(β-manno(oligo)syloxy) ethyl methacrylates (mannosyl-EMA and mannobiosyl-EMA). The chemical structures and radical polymerisations of these novel monomers were determined using 1H and 13C NMR spectroscopy and size-exclusion chromatography. The two new water soluble polymers have a polyacrylate backbone with one or two pendant mannosyl groups per monomeric EMA unit, respectively. These novel glycopolymers may show properties suitable for various technical and biomedical applications responding to the current demand for functional greener materials to replace fossil based ones.

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Wood-Derived Dietary Fibers Promote Beneficial Human Gut Microbiota.

Rosa, S. L. L., Vasiliki, K., Fanny, B., Pope, P. B., Pudlo, N. A., Martens, E. C., Rastall, R. A., et al. (2019). mSphere, 4(1), 1-16.

Woody biomass is a sustainable and virtually unlimited source of hemicellulosic polysaccharides. The predominant hemicelluloses in softwood and hardwood are galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), respectively. Based on the structure similarity with common dietary fibers, GGM and AGX may be postulated to have prebiotic properties, conferring a health benefit on the host through specific modulation of the gut microbiota. In this study, we evaluated the prebiotic potential of acetylated GGM (AcGGM) and highly acetylated AGX (AcAGX) obtained from Norwegian lignocellulosic feedstocks in vitro. In pure culture, both substrates selectively promoted the growth of Bifidobacterium, Lactobacillus, and Bacteroides species in a manner consistent with the presence of genetic loci for the utilization of -manno-oligosaccharides/-mannans and xylo-oligosaccharides/ xylans. The prebiotic potential of AcGGM and AcAGX was further assessed in a pH controlled batch culture fermentation system inoculated with healthy adult human feces. Results were compared with those obtained with a commercial fructooligosaccharide (FOS) mixture. Similarly to FOS, both substrates significantly increased (P < 0.05) the Bifidobacterium population. Other bacterial groups enumerated were unaffected with the exception of an increase in the growth of members of the Bacteroides-Prevotella group, Faecalibacterium prausnitzii, and clostridial cluster IX (P < 0.05). Compared to the other substrates, AcGGM promoted butyrogenic fermentation whereas AcAGX was more propiogenic. Although further in vivo confirmation is necessary, these results demonstrate that both AcGGM and AcAGX from lignocellulosic feedstocks can be used to direct the promotion of beneficial bacteria, thus exhibiting a promising prebiotic ability to improve or restore gut health.

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