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Mixed-linkage (1→3),(1→4)-β-d-glucan (MLG) is a biologically and technologically important hemicellulose, known to occur in three widely separated lineages: the Poales (including grasses and cereals), Equisetum (fern-allies), and some lichens e.g. Iceland moss (Cetraria islandica). Lichenase (E.C. 126.96.36.199) is widely assumed to hydrolyse all (1→4) bonds that immediately follow (1→3) bonds in MLG, generating predominantly the tetrasaccharide β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glc (G4G4G3G; MLG4), the corresponding trisaccharide (G4G3G; MLG3), and sometimes also laminaribiose (G3G; MLG2). The ratio of the oligosaccharides produced characterises each polysaccharide. We report here that digestion of MLG from barley (Hordeum vulgare), Equisetum arvense and C. islandica by Bacillus subtilis lichenase also yields the unexpectedly stable hexasaccharide, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glc (G3G4G4G4G3G, i.e. MLG2-MLG4), identified by thin-layer chromatography, gel-permeation chromatography, HPLC (HPAEC), β-glucosidase digestion, (1)H/(13)C-NMR spectroscopy and mass spectrometry. On HPLC, G3G4G4G4G3G is the major constituent of a peak previously ascribed solely to the nonasaccharide G4G4G4G4G4G4G4G3G. Because it was widely presumed that lichenase would cleave G3G4G4G4G3G to MLG2+MLG4, our data both redefine the substrate specificity of Bacillus lichenase and show previous attempts to characterise MLGs by HPLC of lichenase-digests to be flawed. MLG2 subunits are particularly underestimated; often reported as negligible, they are here shown to be an appreciable constituent of MLGs from all three lineages. We also show that there is no appreciable yield of water-soluble lichenase products with DP>9; potential identities of products previously labelled DP>9 are suggested. Finally, this discovery also provides a opportunity to investigate the spatial distribution of subunits along the MLG chain. We show that MLG2 subunits in barley and Cetraria MLG are not randomly distributed, but predominantly found at the non-reducing end of MLG4 subunits.
|Number of pages||11|
|Publication status||Published - Nov 2013|
- CELL WALL COMPOSITION
- OAT BRAN
- SELECTIVE ENZYMOLYSIS
- EQUISETUM HORSETAILS
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- 1 Finished
SCIBS: Selective chemical intervention in plant cell wall polysaccharide metabolism: consequences for cell expansion
1/08/07 → 31/08/10