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Beta Glucan Toolkit

Megazyme’s complete toolkit for the study of mixed linkage (1,3:1,4)-β-glucan and the enzymes that act on it

Mixed linkage (1,3:1,4)-β-glucan is found in the endosperm cell walls of cereal grains such as oat and barley, and to a lesser extent wheat and rye. The health benefits associated with β-glucan are well established and this makes it a vital component of interest to the food industry. High levels of β-glucan in animal feeds lead to reduced feed intake and decreased feed conversion. 

β-Glucan is also an important analyte in the brewing industry where elevated levels can lead to increased wort viscosity and cause processing problems. It is therefore essential to understand the enzymatic mechanisms for the complete degradation of this polysaccharide, its structure and its functional properties. 


Mixed linkage beta-glucan image

Figure: β-Glucan is composed of D-glucosyl monomeric units linked either β-1,3 or β-1,4.

Assay Kits

Megazyme has developed a novel assay kit for the measurement of mixed linkage β-glucan in all samples (K-BGLU: AOAC 995.16, CODEX Type II method). An assay kit based on the dye-labelled native polysaccharide (K-MBGL) is also available for the measurement of endo-acting β-glucan degrading enzymes including endo-1,3(4)-β-glucanase/lichenase and endo-1,4-β-glucanase/endo-cellulase. endo-1,4-β-Glucanase can be specifically measured using the novel CELLG5 assay kit (K-CELLG5). Free glucose content in a sample can be determined using either glucose oxidase or hexokinase procedures (K-GLUC and K-GLUHK).

Product Code   Product 
K-BGLU  β-Glucan (β-1,3:1,4 mixed linkage)                                         
K-MBGL β-Glucanase (malt and microbial) 
K-CELLG5 Cellulase/endo-1,4-β-glucanase (CELLG5 method)                                                                              
K-CELLG3  Cellulase Assay Kit (CELLG3 Method) (useful in distinguishing different types of endo-cellulase based on substrate subsite binding requirements)
K-GLUC  D-Glucose (GOPOD format)
K-GLUHK  D-Glucose (HK format)  

Carbohydrates

Oligosaccharides

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.

Product Code    Product                                                                                                       
O-BGTRIA  1,3:1,4-β-Glucotriose (A)  1,3:1,4-beta-glucotriose A   
O-BGTRIB
1,3:1,4-β-Glucotriose (B)
 1,3:1,4-beta-glucotriose B
O-BGTETB  1,3:1,4-β-Glucotetraose (B) 1,3:1,4-beta-glucotetraose 
O-BGTETC  1,3:1,4-β-Glucotetraose (C)        1,3:1,4-beta-glucotetraose C
O-CTR
Cellotriose
 Cello-oligosaccharides
O-CTE
Cellotetraose 
O-CPE  Cellopentaose
O-CHE  Cellohexaose 

Polysaccharides

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.

Product Code   Product
P-BGBL  β-Glucan (barley; low viscosity, high purity) 
P-BGBM
β-Glucan (barley; medium viscosity, high purity)                                                                                                     
P-BGBH  β-Glucan (barley; high viscosity, high purity) 
P-BGOM  β-Glucan (oat; medium viscosity, high purity) 
P-BGOH  β-Glucan (oat; high viscosity, high purity) 
P-BGCFA  β-Glucan CFA standard 
P-MWBGS  β-Glucan MW standards
P-LICHN Lichenan (Icelandic moss) 
Enzymes

Lichenase (EC 3.2.1.73) 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 3.2.1.4) 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 3.2.1.21) 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 3.2.1.91) act in a similar exo- fashion but cleave disaccharide (cellobiosyl) units.

beta glucan enzymes

Figure: Schematic representation of the hydrolytic action of β-glucan degrading enzymes.

Product Code   Product      
 endo-1,3(4)-β-Glucanases 
 E-LICHN Lichenase (Bacillus sp.)
 E-LICACT RecNon-specific endo-β-Glucanase (C. thermocellum)                                                                       
 endo-1,4-β-Glucanases
E-CELAN endo-Cellulase (A. niger) (pH optima 4.5)
E-CELBA  Recendo-Cellulase (B. amyloliquefaciens) (pH optima 6.0; thermostable)
E-CELTE  endo-Cellulase (T. emersonii) (pH optima 4.5)
E-CELTR  endo-Cellulase (T. longibrachiatum) (pH optima 4.5; hydrolyses xyloglucan)
E-CELTM  endo-Cellulase (T. maritima) (pH optima 6.0; thermostable)
exo-1,4-β-Glucosidases
E-BGLUC β-Glucosidase (A. niger) (pH optima 4.0)
E-BGOSAG β-Glucosidase (Agrobacterium sp.) (pH optima 6.5)
E-BGOSPC
β-Glucosidase (thermostable) (P. chrysosporium) (pH optima 5.0; thermostable)
E-BGOSTM
β-Glucosidase (thermostable) (T. maritima) (pH optima 6.5; thermostable)
exo-1,3-β-Glucanases
E-EXG5AO  Recexo-1,3-β-Glucanase (Aspergillus oryzae) (pH optima 5.0)
E-EXBGOS  exo-1,3-β-D-Glucanase + β-Glucosidase 
E-EXBGTV  Recexo-1,3-β-Glucanase (Trichoderma virens) (pH optima 4.5)
Cellobiohydrolases 
E-CBHI Cellobiohydrolase I (T. longibrachiatum)
E-CBHIIM  RecCellobiohydrolase II (microbial)
Rec(recombinant enzyme)

Enzyme Substrates

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.

Product Code Product Name Chemical Structure 
endo-(1,3:1,4)-β-Glucanase/
Lichenase substrates

O-CNPBG3 2-Chloro-4-nitrophenyl-
β-(1,3:1,4)-
glucotrioside
NEW 
O-CNPBG3 and 4
O-CNPBG4  2-Chloro-4-nitrophenyl-
β-(1,3:1,4)-glucotetraoside
NEW 
O-4MUBG3  4-Methylumbelliferyl-
β-(1,3:1,4)-
glucotrioside
NEW 
 4-Methylumbelliferyl- beta-1,3:1,4-glucotrioside
endo-(1,4)-β-Glucanase/
endo-Cellulase substrates

O-CPNPG2-100 2-Chloro-4-nitrophenyl-
β-cellobioside 
2-Chloro-4-nitrophenyl-beta-cellobioside
O-CNPBG3
2-Chloro-4-nitrophenyl-
β-cellotrioside

O-CPNPG4-50  2-Chloro-4-nitrophenyl-
β-cellotetraoside
O-CPNPG5
2-Chloro-4-nitrophenyl-
β-cellopentaoside

exo-1,4-β-Glucosidase substrates
O-PNPBG  4-Nitrophenyl-
β-D-glucopyranoside
4-Nitrophenyl-beta-D-glucopyranoside



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.

Soluble substrate overview

Figure: Schematic representation of the use of dyed polysaccharides (soluble) for the assay of endo-β-glucanase activity.

Product Code   Product 
endo-(1,3:1,4)-β-Glucanase substrates (and endo-(1,4)-β-Glucanase substrates)
Tablets 
T-BGZ  β-Glucazyme  (containing AZCL-Barley β-glucan)
Soluble Substrate 
S-ABG100 Azo-Barley Glucan
Insoluble Substrate 
I-AZBGL  AZCL-Barley β-Glucan 
 
Specific endo-1-4-β-Glucanase substrates
Tablets
T-CAF  Cellazyme AF (containing AZCL-HP-cellulose)
T-CCZ
Cellazyme C (containing AZCL-HP-cellulose)
T-CTZ  Cellazyme T (containing AZCL-tamarind xyloglucan)
Soluble Substrates 
S-ACMC  Azo-CM-Cellulose (powder) 
S-ACMCL Azo-CM-Cellulose (liquid) 
S-AZXG Azo-Xyloglucan (Tamarind)                                                                                                                      
Insoluble Substrates  
I-AZCEL  AZCL-HE-Cellulose 
I-AZXYG  AZCL-Xyloglucan (Tamarind)
I-ACELL  Azo-α-Cellulose
I-AAVIC  Azo-Avicel