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Mannopentaose

Mannopentaose O-MPE
Product code: O-MPE
€134.00

30 mg

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Content: 30 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 70281-35-5
Molecular Formula: C30H52O26
Molecular Weight: 828.7
Purity: > 98%
Substrate For (Enzyme): endo-1,4-β-Mannanase

High purity Mannopentaose 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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Efficient and green production of manno-oligosaccharides from Gleditsia microphylla galactomannans using CO2 and solid acid in subcritical water.

Xu, W., Han, M., Zhang, W., Tang, M., Zhang, F. & Jiang, J. (2022). LWT, 156, 113019.

This study aimed to produce manno-oligosaccharides (MOS) from Gleditsia microphylla galactomannans (GMG) using CO2 and solid acid (Amberlyst-35) in subcritical water. The optimal condition for MOS preparation was 3 MPa CO2, 0.1 g/g solid acid (relative to GMG) at 150°C for 40 min. The maximum MOS yield with a degree of polymerization from 2 to 4 (M2-M4) was 52.19%, which doubled the yield of MOS compared to either using solely CO2 or solid acid. Solid acid showed excellent performance in producing MOS under subcritical H2O-CO2 condition, due to the enhanced mass transfer efficiency and increased H+ concentration in the reaction system. The solid acid can be easily separated and reused. Comparing with traditional methods used to produce MOS, this approach has many merits such as higher galactomannan hydrolysis efficiency (largely reduced time and higher MOS yield), purer M2-M4 product, lower costs, and more environmental-friendly.

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Transglycosylation by β-mannanase TrMan5A variants and enzyme synergy for synthesis of allyl glycosides from galactomannan.

Butler, S. J., Birgersson, S., Wiemann, M., Arcos-Hernandez, M. & Stålbrand, H. (2021). Process Biochemistry, 112, 154-166.

Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0-5.3 and showed >80% remaining activity after incubation at 40°C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH2 column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1-3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.

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Fast and specific peroxygenase reactions catalyzed by fungal mono-copper enzymes.

Rieder, L., Stepnov, A. A., Sørlie, M. & Eijsink, V. G. (2021). Biochemistry, In Press.

The copper-dependent lytic polysaccharide monooxygenases (LPMOs) are receiving attention because of their role in the degradation of recalcitrant biomass and their intriguing catalytic properties. The fundamentals of LPMO catalysis remain somewhat enigmatic as the LPMO reaction is affected by a multitude of LPMO- and co-substrate-mediated (side) reactions that result in a complex reaction network. We have performed kinetic studies with two LPMOs that are active on soluble substrates, NcAA9C and LsAA9A, using various reductants typically employed for LPMO activation. Studies with NcAA9C under “monooxygenase” conditions showed that the impact of the reductant on catalytic activity is correlated with the hydrogen peroxide-generating ability of the LPMO-reductant combination, supporting the idea that a peroxygenase reaction is taking place. Indeed, the apparent monooxygenase reaction could be inhibited by a competing H2O2-consuming enzyme. Interestingly, these fungal AA9-type LPMOs were found to have higher oxidase activity than bacterial AA10-type LPMOs. Kinetic analysis of the peroxygenase activity of NcAA9C on cellopentaose revealed a fast stoichiometric conversion of high amounts of H2O2 to oxidized carbohydrate products. A kcat value of 124 ± 27 s–1 at 4 °C is 20 times higher than a previously described kcat for peroxygenase activity on an insoluble substrate (at 25 °C) and some 4 orders of magnitude higher than typical “monooxygenase” rates. Similar studies with LsAA9A revealed differences between the two enzymes but confirmed fast and specific peroxygenase activity. These results show that the catalytic site arrangement of LPMOs provides a unique scaffold for highly efficient copper redox catalysis.

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Production and in vitro evaluation of prebiotic manno-oligosaccharides prepared with a recombinant Aspergillus niger endo-mannanase, Man26A.

Magengelele, M., Hlalukana, N., Malgas, S., Rose, S. H., van Zyl, W. H. & Pletschke, B. I. (2021). Enzyme and Microbial Technology, 150, 109893.

In this study, a GH26 endo-mannanase (Man26A) from an Aspergillus niger ATCC 10864 strain, with a molecular mass of 47.8 kDa, was cloned in a yBBH1 vector and expressed in Saccharomyces cerevisiae Y294 strain cells. Upon fractionation by ultra-filtration, the substrate specificity and substrate degradation pattern of the endo-mannanase (Man26A) were investigated using ivory nut linear mannan and two galactomannan substrates with varying amounts of galactosyl substitutions, guar gum and locust bean gum. Man26A exhibited substrate specificity in the order: locust bean gum ≥ ivory nut mannan > guar gum; however, the enzyme generated more manno-oligosaccharides (MOS) from the galactomannans than from linear mannan during extended periods of mannan hydrolysis. MOS with a DP of 2–4 were the major products from mannan substrate hydrolysis, while guar gum also generated higher DP length MOS. All the Man26A generated MOS significantly improved the growth (approximately 3-fold) of the probiotic bacterial strains Streptococcus thermophilus and Bacillus subtilis in M9 minimal medium. Ivory nut mannan and locust bean gum derived MOS did not influence the auto-aggregation ability of the bacteria, while the guar gum derived MOS led to a 50 % reduction in bacterial auto-aggregation. On the other hand, all the MOS significantly improved bacterial biofilm formation (approximately 3-fold). This study suggests that the prebiotic characteristics exhibited by MOS may be dependent on their primary structure, i.e. galactose substitution and DP. Furthermore, the data suggests that the enzyme-generated MOS may be useful as potent additives to dietary foods.

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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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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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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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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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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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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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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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