Role of (1,3)(1,4) β-glucan in cell walls: Interaction with cellulose.
Kiemle, S. N., Zhang, X., Esker, A. R., Toriz, G., Gatenholm, P. & Cosgrove, D. J. (2014). Biomacromolecules, 15(5), 1727-1736.
(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.
endo-β-1,4-Mannanases from blue mussel, Mytilus edulis: purification, characterization, and mode of action.
Xu, B., Hägglund, P., Stålbrand, H. & Janson, J. C. (2002). Journal of Biotechnology, 92(3), 267-277.
Two variants of an endo-β-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39 216 and 39 265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50–55°C. Their stability decreases rapidly when going from 40 to 50°C. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan.
Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis.
Westphal, Y., Schols, H. A., Voragen, A. G. J. & Gruppen, H. (2010). Journal of Chromatography A, 1217(5), 689-695.
Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSn-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar β-(1,4)-xylo- and β-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of β-(1,4)-galacto-, β-(1,4)-manno- and α-(1,4)-gluco-oligosaccharides. Charged oligomers such as α-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.
Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase.
Mizutani, K., Fernandes, V. O., Karita, S., Luís, A. S., Sakka, M., Kimura, T., Jackson, A., Zhang, X., Fontes, C. M. G. A., Gilbert, H. J. & Sakka, K. (2012). Applied and Environmental Microbiology, 78(14), 4781-4787.
In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.
A non-modular endo-β-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa.
Braithwaite, K. L., Black, G. W., Hazlewood, G. P., Ali, B. R. S. & Gilbert, H. J. (1995). Biochem. J, 305, 1005-1010.
Pseudomonas fluorescens subsp. cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide. To isolate gene(s) from P. fluorescens subsp. cellulosa encoding endo-β-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan. The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of Mr 46,938. The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide. Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity. Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases. These data indicate that the mannanase is non-modulator, comprising a single catalytic domain. Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp. Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases. Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall. The enzyme had a pH optimum of approx. 7.0, was resistant to proteolysis and had an Mr of 46,000 when expressed by Escherichia coli.
Restricted access of proteins to mannan polysaccharides in intact plant cell walls.
Marcus, S. E., Blake, A. W., Benians, T. A. S, Lee, K. J., Poyser, C., Donaldson, L., Leroux, O., Rogowski, A., Petersen, H. L., Boraston, A., Gilbert, H. J., Willats, W. G. T. & Paul Knox, J. (2010). The Plant Journal, 64(2), 191-203.
How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.
A carbohydrate binding module as a diversity‐carrying scaffold.
Gunnarsson, L. C., Karlsson, E. N., Albrekt, A. -S., Andersson, M., Holst, O. & Ohlin, M. (2004). Protein Engineering, Design & Selection, 17(3), 213-221.
The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large‐scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4‐2, from a xylanase found in Rhodothermus marinus, as a diversity‐carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4‐2. Despite the small size of the library (1.6×106 clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 ± 5.3°C). All our results demonstrate that the CBM4‐2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
Purification and some properties of a thermostable acidic endo‐β‐1,4‐D‐mannanase from Sclerotium (Athelia) rolfsii.
Sachslehner, A. & Haltrich, D. (1999). FEMS Microbiology Letters, 177(1), 47-55.
The phytopathogenic fungus Sclerotium (Athelia) rolfsii forms one major endo-β-1,4-D-mannanase (EC 18.104.22.168) under non-induced and derepressed conditions, i.e. after depletion of glucose which was used as the only carbohydrate substrate for its cultivation. This mannanase was purified to electrophoretic homogeneity by ammonium sulfate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and gel filtration. The enzyme is a glycoprotein with a molecular mass of 46.5±2 kDa (SDS-PAGE), an isoelectric point of 2.75, and a pH optimum of 3.0–3.5. The enzyme is especially stable in the acidic region with an exceptional half-life of activity of 41 days at pH 4.5 and 50°C. It exerts activity on β-1,4-mannan from ivory nut, which is hydrolyzed mainly to mannobiose and mannotriose, as well as on glucomannan, galactomannan, galactoglucomannan, and mannooligosaccharides not smaller than mannotetraose. The main end-products mannotriose and to a lesser extent mannobiose inhibit its activity moderately.
X4 modules represent a new family of carbohydrate-binding modules that display novel properties.
Bolam, D. N., Xie, H., Pell, G., Hogg, D., Galbraith, G., Henrissat, B. & Gilbert, H. J. (2004). Journal of Biological Chemistry, 279(22), 22953-22963.
The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.
Digestion of single crystals of mannan I by an endo‐mannanase from Trichoderma reesei.
Sabini, E., Wilson, K. S., Siika‐aho, M., Boisset, C. & Chanzy, H. (2000). European Journal of Biochemistry, 267(8), 2340-2344.
The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a β-mannanase (EC 22.214.171.124 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.