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Mannohexaose

Mannohexaose O-MHE
Product code: O-MHE
€126.00

20 mg

Prices exclude VAT

Available for shipping

Content: 20 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 70281-36-6
Molecular Formula: C36H62O31
Molecular Weight: 990.9
Purity: > 95%
Substrate For (Enzyme): endo-1,4-β-Mannanase

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

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Publications
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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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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High-resolution structure of a modular hyperthermostable endo-β-1, 4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain.

da Silva, V. M., Cabral, A. D., Sperança, M. A., Squina, F. M., Muniz, J. R. C., Martin, L., Nicolet, Y. & Garcia, W. (2020). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 140437.

The endo-β-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-β-1,4-mannanase structure determined. The structure exhibits two domains, a (β/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like β-sandwich fold formed of seven β-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.

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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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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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Evolutionary origin of O‐acetyltransferases responsible for glucomannan acetylation in land plants.

Zhong, R., Cui, D., & Ye, Z. H. (2019). New Phytologist, 224(1), 466-479.

Mannans are an abundant cell wall polysaccharide in bryophytes, seedless vascular plants and gymnosperms. A previous study has shown that mannan acetylation in Arabidopsis and konjac is mediated by mannan O‐acetyltransferases belonging to the Domain of Unknown Function (DUF) 231 family. However, little is known about the acetylation patterns of mannans in bryophytes and seedless vascular plants, and the evolutionary origin of mannan O‐acetyltransferases in land plants has not yet been studied. Phylogenetic analysis of the DUF231 family revealed that DUF231 members were present in the charophycean green algae and evolved to form overlapped and divergent phylogenetic groups in different taxa of land plants. Acetyltransferase activity assays of recombinant proteins demonstrated that a number of group II DUF231 members from moss, Selaginella, pine, spruce, rice and poplar were mannan 2‐O‐ and 3‐O‐acetyltransferases, whereas the two group I DUF231 members from the alga Klebsormidium nitens were not. Structural analysis of mannans from moss and Selaginella showed they were composed of mannosyl and glucosyl residues and the mannosyl residues were acetylated at O‐2 and O‐3. These findings indicate that although the DUF231 genes originated in algae, their recruitment as mannan O‐acetyltransferases probably occurred in bryophytes, and the biochemical functions of these O‐acetyltransferases are evolutionarily conserved throughout land plants.

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

Kaira, G. S. & Kapoor, M. (2019). Biochemical and Biophysical Research Communications, 510(3), 358-363.

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.

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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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Enzymatic hydrolysis of Hutan Jati Variety Cultivar Tacca (Tacca Leontopetaloides) Starch by the Brevibacterium sp. α-Amylase and Its Potential for Production of Maltooligosaccharides.

Rahmani, N., Putri, F. H., Martin, A. F. & Yopi, Y. (2019). Biotropia, 26(2), 272129.

The aim of research are extraction and physico-chemical characterization, determination of the optimum conditions for enzymatic hydrolysis of starch from Tacca tuber to produce maltooligosaccharides, and analysize of character of the product. The analysis of the product is conducted by calculating the amount of reducing sugar, and total sugar, degree of polymerization, TLC (Thin Layer Chromatography) analysis, as well as HPLC (High Performance Liquid Chromatography) analysis. Hutan Jati variety cultivar Tacca was selected from three Tacca variety cultivars (Hutan Jati, Pulau Katang and Gunung Batur) to produce maltooligosaccharides by enzymatic hydrolysis of crude Brevibacterium sp.α-amilase. The optimum conditions of enzymatic hydrolysis of Hutan Jati variety cultivar Tacca starch for production of maltooligosaccharides was obtained at a substrate concentration of 3% (w/v), the ratio of enzyme and substrate of 1:5 at 6h incubation time. It was obtained 34.4903 grams of powder maltooligosaccharide from 250 mL of fresh hydrolizate. The results of TLC and HPLC analysis showed similar yield to both of the liquid and the powder maltooligosaccharides with maltose, maltotriose, and maltotetraose as main product. Considering the result of psycho-chemically characteristic and the product of maltooligosaccharides from Hutan Jati variety cultivar Tacca tuber starch, presented in this work, may be considered as a potential, strong candidates for future applications as a source of maltooligosaccharide production, especially maltotriose and maltotetraose in functional food industry.

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Development of an advanced integrative process to create valuable biosugars including manno-oligosaccharides and mannose from spent coffee grounds.

Nguyen, Q. A., Cho, E. J., Lee, D. S. & Bae, H. J. (2019). Bioresource Technology, 272, 209-216.

Spent coffee grounds (SCG) or coffee residue wastes (CRW) provide excellent raw material for mannose and bioethanol production. In this study, SCG were used to produce valuable biosugars, including oligosaccharides (OSs), manno-oligosaccharides (MOSs), mannose, and bioethanol. SCG were subjected to delignification and defatting, producing SCG-derived polysaccharides. Two-stage enzymatic hydrolysis (short- and long-term) was performed to produce short-chain manno-oligosaccharides (MOSs) and monosaccharides (MSs), respectively. From 100 g dry weight (DW) amounts of SCG, approximately 77 g delignified SCG and 61 g SCG-derived polysaccharides, amounts of 15.9 g of first biosugars (mostly MOSs), 25.6 g of second biosugars (mostly MSs), and 3.1 g of bioethanol, were recovered. This technique may aid in the production of high-value mannose and OSs from SCG and other lignocellulosic biomasses that contain specific polysaccharides.

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Production of mannose-containing hydrolysates and investigation of their ability to normalize the microflora composition of the gastrointestinal tract of poultry.

Korneeva, O., Anokhina, E., Shuvayeva, G., Isuva, M. & Startseva, S. (2018). International Multidisciplinary Scientific GeoConference: SGEM, 18(6.2), 475-482.

One of the ways to maintain the normal gastrointestinal tract microflora of poultry is to use prebiotic supplements in their feed. Among the prebiotics used in poultry farming, there are additives based on mannooligosaccharides from the cell walls of the yeast Saccharomyces cerevisiae. There are no similar domestic prebiotic drugs available, which determines the necessity and relevance of their development. For the production of mannose-containing hydrolysates, there was studied the process of hydrolysis of mannans of plant raw materials under the influence of the enzyme preparation of Trichoderma harzianum β-mannanase. The production of mannose-containing hydrolysates with the maximum degree of mannans splitting was provided by the dosage of the 15 U/g substrate enzyme and the duration of the process for 4 hours. Qualitative and quantitative analysis of the produced hydrolysates revealed the presence of mannose, mannobiose, mannotriose, mannotetrose, mannohexoses in the amount of, in mg / ml: 29.1, 22.1, 42.2, 19.1 and 4.3, respectively. Studying the prebiotic properties of mannose-containing hydrolysates in vitro showed that the maximum accumulation of Bifidobacterium bifidum biomass on medium with these carbohydrates was observed by the 42nd h of cultivation and correlated with the accumulation of organic acids in the medium. Mannose-containing hydrolysates stimulated the development of B. bifidum to a greater extent than mannose and did not rank below to the known commercial prebiotic - inulin. The study of the effect of mannose-containing hydrolysates in the amount from 0.1 to 1% to the feed weight on the gastrointestinal microflora of chickens with experimental dysbiosis showed that an increase in the number of bifidobacteria and lactic acid bacteria was observed already on the 5th day of the experiment in all the doses tested. Complete normalization of the gastrointestinal microflora composition of poultry was observed when mannose-containing hydrolysates were introduced in the amount of 1% to the feed weight on the 10th day.

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Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10.

Zhou, C., Xue, Y. & Ma, Y. (2018). Microbial Cell Factories, 17(1), 124.

Background: β-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. Results: A thermo-alkaline β-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 C with good stability at pH 7.0-11.5 and below 80 C. BcManA demonstrated high cleavage capability on polysaccharides containing β-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg−1 was observed on konjac glucomannan with the Kmand kcat value of 0.62 g l−1 and 1238.9 s−1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml−1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml−1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. Conclusions: The thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml−1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline β-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other β-mannanases in B. subtilis.

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Publication

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

Zhong, R., Cui, D. & Ye, Z. H. (2018). Planta, 1-13.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

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