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.
Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase.
Ademark, P., Varga, A., Medve, J., Harjunpää, V., Drakenberg, T., Tjerneld, F. & Stålbrand, H. (1998). Journal of Biotechnology, 63(3), 199-210.
The enzymes needed for galactomannan hydrolysis, i.e. β-mannanase, α-galactosidase and β-mannosidase, were produced by the filamentous fungus Aspergillus niger. The β-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the β-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger β-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus β-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.
Protein release from galactoglucomannan hydrogels: influence of substitutions and enzymatic hydrolysis by β-mannanase.
Roos, A. A., Edlund, U., Sjoberg, J., Albertsson, A. C. & Stålbrand, H. (2008). Biomacromolecules, 9(8), 2104-2110.
O-Acetyl-galactoglucomannan (AcGGM) is the major soft-wood hemicellulose. Structurally modified AcGGM and hydrogels of AcGGM were prepared. The degree of substitution (DS) of AcGGM was modified enzymatically with α-galactosidase, and chemically with an acrylate derivative, 2-hydroxyethylmethacrylate (HEMA). The hydrolysis of AcGGM with β-mannanase was shown to increase with decreasing DS. AcGGM hydrogels were prepared from chemically modified AcGGM with varying DS of HEMA. Bovine serum albumin (BSA) was encapsulated in hydrogels. A spontaneous burst release of BSA was decreased with increased DS of HEMA. The addition of β-mannanase significantly enhanced the BSA release from hydrogels with a DS of 0.36, reaching a maximum of 95% released BSA after eight hours compared to 60% without enzyme. Thus, both the pendant group composition and the enzyme action are valuable tools in the tailoring of hydrogel release profiles of potential interest for intestine drug delivery.
Mannotriose regulates learning and memory signal transduction in the hippocampus.
Zhang, L., Dai, W., Zhang, X., Gong, Z. & Jin, G. (2013). Neural Regeneration Research, 8(32), 3020-3026.
Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Primary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10-4 mol/L) corticosterone, and treated with 1 × 10-4 mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorticoid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.
Operational and storage stability of neutral β-mannanase from Bacillus licheniformis.
Zhang, J., He, M. & He, Z. (2002). Biotechnology Letters, 24(19), 1611-1613.
The stability of neutral β-mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l-1 over 30 min at 25°C. In an enzymatic membrane reactor operated at 50°C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4°C after one year.
Substrate Specificities of α-Galactosidase from Rice.
Li, S. H., Zhu, M. P. & Li, T. P. (2011). Advanced Materials Research, 183, 447-451.
The α-galactosidase from rice cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose and stachyose, but also liberated the terminal galactosyl residues attached O-6 position of the reducing-end mannosyl residue in mannobiose and mannotriose. In addition, the enzyme tore off the stubbed galactosyl residues attached inner-mannosyl residues in mannopentaose. It also could catalyze efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.
Purification and characterization of two β-mannanases from Trichoderma reesei.
Stålbrand, H., Siika-aho, M., Tenkanen, M. & Viikari, L. (1993). Journal of Biotechnology, 29(3), 229-242.
Five enzymes with mannanase activity were separated from Trichoderma reesei culture filtrate using analytical isoelectric focusing and subsequently detected with the zymogram technique. The crude enzymes had isoelectric points in the range of 3.6–6.5. Two of the mannanases with pI values of 4.6 and 5.4 were purified using ion-exchange chromatography, affinity chromatography and chromatofocusing. The molecular weights determined with SDS-PAGE were 51 000 (mannanase pI 4.6) and 53 000 (mannanase pI 5.4). The two enzymes had similar properties with respect to pH optimae and pH stabilities. Both mannanases hydrolyzed ivory nut mannan mainly to mannotriose and mannobiose. The specific activities (against locust bean gum) of the purified enzymes were 1860 and 1430 nkat mg-1 for the pI 4.6 and pI 5.4 mannanases, respectively.
Fractionation of extracted hemicellulosic saccharides from Pinus pinaster wood by multistep membrane processing.
González-Muñoz, M. J., Rivas, S., Santos, V. & Parajó, J. C. (2013). Journal of Membrane Science, 428, 281-289.
Hemicelluloses of Pinus pinaster wood were selectively separated from cellulose and lignin by reaction with hot, compressed water (autohydrolysis) under optimized conditions. The reaction liquor contained polymeric or oligomeric hemicellulose saccharides (POHS, accounting jointly for 69.6% of the dissolved wood fraction), followed by monosaccharides (accounting for 20.0% of the non-volatile compounds), and non-saccharide compounds. For concentration, purification and fractionation purposes, liquors from hydrothermal processing were subjected to consecutive steps of diafiltration and concentration using membranes of 10, 5, 3, 1 and 0.3 kDa molar mass cut-off. Samples from selected process streams were characterized by chromatographic and spectrometric methods. The experimental results provided information on the separation and refining effects achieved by the various membrane processing steps, which affect the technological properties of products.
β-Mannanolytic system of Aureobasidium pullulans.
Kremnický, L. & Biely, P. (1997). Archives of Microbiology, 167(6), 350-355.
A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-D-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase.
The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.
Hogg, D., Pell, G., Dupree, P., Goubet, F., Martin-Orue, S., Armand, S. & Gilbert, H. (2003). Biochem. J, 371(3), 1027-1043.
β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.