A number of mixed linkage (β-1,3:1,4) DP3 and DP4 gluco-oligosaccharides have been prepared with high purity for use as chromatographic standards and for use in enzyme characterisation and binding studies.The corresponding cello-oligosaccharides may also prove useful in this regard.
Megazyme produces and supplies high purity (1,3:1,4)-β-D-glucan from oat and barley.
β-Glucans which have high, medium and low viscosity grades have been prepared with these viscosities indicating the molecular weight range in each product. Lichenan isolated from Icelandic moss is an alternative source of β-glucan with a higher proportion of β-1,3 linkages. Carboxymethyl cellulose 4 M is a medium viscosity cellulose with a degree of substitution with carboxymethyl groups of 0.4. This lower degree of substitution than carboxymethyl cellulose offered by other suppliers (typically DS 0.7) is more readily hydrolysed by the full spectrum of endo-cellulases, and thus universally useful for the assay of this enzyme.
Lichenase (EC 22.214.171.124) selectively hydrolyses β-1,4 glycosidic bonds adjacent to a β-1,3 linked glucose residue on their reducing side. endo-Cellulases (endo-1,4-β-glucanase) (EC 126.96.36.199) hydrolyse the β-1,4 glycosidic bonds present in the polysaccharide backbone. It cleaves the 1,4-β-D-glucosidic linkage on the non-reducing side of a 1,3-β-linked D-glucosyl residue, but not that on the reducing side. It also produces various cello-oligosaccharides on hydrolysis of the long stretches of 1,4-β-D-glucosyl chains in these glucans.
β-Glucosidases (EC 188.8.131.52) act sequentially, hydrolysing one glucose residue at a time, from the non-reducing end of oligosaccharide/polysaccharide fragments. They have varying specificity for β-1,4 and β-1,3 glycosidic bonds. Cellobiohydrolases (EC 184.108.40.206) act in a similar exo- fashion but cleave disaccharide (cellobiosyl) units.
Figure: Schematic representation of the hydrolytic action of β-glucan degrading enzymes.
Colourimetric oligosaccharides with a chemically-defined structure have been prepared and are suitable for use in both manual and high throughput automated analysis applications. The activity of the enzyme assayed using water soluble nitrophenyl-linked colourimetric oligosaccharides can be determined directly by measuring the absorption increase at 400 nm which is directly proportional to the release of the nitrophenyl colourimetric group in solution. Similar procedures are used for fluorometric substrates which generally provide higher sensitivity. The fluorescence of the 4-methylumbelliferyl group released by the action of the enzyme being tested is measured and correlates to the amount of enzyme activity in the reaction. Three lichenase substrates have recently been developed while the colourimetric cello-oligosaccharides and 4-nitrophenyl-β-D-glucopyranoside may also prove useful as substrates for endo-1,4-β-glucanase and exo-1,4-β-glucosidase respectively.
Insoluble and soluble chromogenic enzyme substrates for the detection and measurement of endo-(1,3:1,4)-β-glucanase activity have been prepared from (1,3:1,4)-β-glucan. These substrates produce a colourimetric response upon incubation with a suitable endo-(1,3:1,4)-β-glucanase that can be related to enzyme activity through the standard curve provided (S-ABG100 only) or be used for screening on agar plates. The insoluble substrate is also available in tablet form to increase accuracy and convenience for the user when assaying specific enzyme activity. It should be noted that as many endo-1,4-β-glucanases also act on (1,3:1,4)-β-glucan, these substrates will also be hydrolysed by some endo-1,4-β-glucanases. Analogous versions of these dyed polysaccharide substrates that are specific for endo-1,4-β-glucanase are also available.
Figure: Schematic representation of the use of dyed polysaccharides (soluble) for the assay of endo-β-glucanase activity.