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Arabinoxylan Toolkit

Megazyme’s complete solution for the study of arabinoxylan and the enzymes that act on it

Arabinoxylan is a significant component of cereal hemicelluloses. It has become a functional food ingredient of interest with beneficial health benefits linked to its prebiotic effects. Understanding the enzymatic mechanisms for the complete degradation of this polysaccharide is also essential for the biofuels industry by allowing the efficient conversion and utilisation of plant biomass. An article recently published by McClearyet al. discusses the action of the various enzymes involved in the hydrolysis of wheat arabinoxylan.

Structurally, arabinoxylan consists of a β-1,4 linked xylan backbone with the xylose monomers bearing a mixture of either 3- monosubstituted or 2,3-disubstituted α-L-arabinose. Some arabinofuranosyle residues are further substituted with ferulic acid, which under certain circumstances can crosslink to other ferulic acid residues, forming di-ferulate bridges.

Arabinoxylan

Figure: Arabinoxylan contains D-xylosyl monomeric units linked β-1,4 and extensively modified with α-L-arabinofuranosyl residues on positions 2 and 3.

Assay Kits

Megazyme currently provides kits for the assay of xylanase activity and is developing an enzymatic assay kit for the determination of wheat flour arabinoxylan. This will be the first commercially available kit of its kind. 

Product Code  Product Name                                                                                                                                      
K-XYLOSE      Xylose (can be used for the measurement Wheat Flour Arabinoxylan)                                                   
K-XYLS Xylanase (Xylazyme AX) 
K-AZOWAX  
Xylanase (Azo-Wax)                                                                                                                                  

Carbohydrates

Oligosaccharides
Specific xylo-oligosaccharides (XOS) and arabino-xylooligosaccharides (AXOS) have been prepared with high purity for use, as chromatographic standards, and in enzyme characterisation and binding studies.

Product Code Product Name               Chemical Structure                                               
 1,4-β-D-Xylo-oligosaccharides
O-XBI  Xylobiose                              Xylo-oligosaccharides      
O-XTR Xylotriose
O-XTE  Xylotetraose
O-XPE  Xylopentaose
O-XHE  Xylohexaose
 Arabinoxylo-oligosaccharides (AXOS)
O-A3X  32-α-L-Arabinofuranosyl
-xylobiose (A3X)      
 Arabinofuranosyl-xylotriose (A2XX + A3XX)
O-A2XX  23-α-L-Arabinofuranosyl
-xylotriose  (A2XX)     
Arabinofuranosyl-xylotriose
O-XA3XX  33-α-L-Arabinofuranosyl
-xylotetraose (XA3XX ) 
Arabinofuranosyl-xylotetraose
O-XAXXMIX 33-α-L- plus 23-α-L-
Arabinofuranosyl-
xylotetraose  
(XA3XX/XA2XX)
 mixture                
  
XAXXMIX

Polysaccharides

Arabinoxylan polysaccharides obtained from rye and wheat are available from Megazyme. High, medium and low viscosity grades have been prepared with these viscosities indicating the molecular weight range of each product. Acid and enzyme debranched arabinoxylans have also been produced providing alternative substrates with varying degrees of arabinose substitution. Xylan sourced from beechwood is available as a substrate for reducing sugar assays of endo-1,4-β-xylanase. Beechwood xylan contains approximately 10% 4-O-methylglucuronate substitution, which is exactly the same as that in birchwood xylan.

Product Code Product Name               
P-RAXY Arabinoxylan (rye flout; high viscosity)                                           
P-WAXYL  Arabinoxylan (wheat flour; low viscosity) 
P-WAXYM  Arabinoxylan (wheat flour; medium viscosity) 
P-WAXYH  Arabinoxylan (wheat flour; high viscosity) 
P-WAXYI  Arabinoxylan (wheat flour; insoluble) 
P-ADWAX22  Arabinoxylan (acid debranched; 22% ara)NEW 
P-ADWAX26  Arabinoxylan (acid debranched; 26% ara)NEW
P-EDWAX30       
Arabinoxylan (enzymically debranched using an α-L-arabinofuranosidase
that removes 
1,3-linked α-L-arabinofuranose from doubly substituted
D-xylose residues, 30% ara)
NEW
 P-XYLNBE Highly purified xylan from beechwood. Suitable as a replacement for
birchwood xylan as a substrate for β-xylanase in DNSA
reducing sugar assays
NEW               
P-WAXYNS   Arabinoxylan (wheat flour; low viscosity;
specifically for use in Nelson-Somogyi reducing sugar assays for β-xylanase)
COMING SOON                       

Enzymes

endo
-1,4-β-Xylanases (EC 3.2.1.8) have the ability to hydrolyse the interlinking β-1,4 glycosidic bonds present in the xylan backbone to varying degrees based on 1) the identity of the endo-xylanase in question and 2) the neighbouring arabinose substitution present.

exo-1,4-β-Xylosidases (EC 3.2.1.37) sequentially hydrolyse xylose residues, one unit at a time, from the non-reducing end of xylo-oligosaccharides.

α-L-Arabinofuranosidases (EC 3.2.1.55) can cleave the arabinofuranose residues from the xylan backbone with strict specificity in some cases for 3-mono-substituted or 2,3-di-substituted arabinofuranose bearing xylose residues.

Feruloyl esterases (EC 3.1.1.73) cleave the ferrulate crosslinks between arabinoxylan and other polymeric structures within hemicellulose.

Arabinoxlan Enzymes image

Figure:
Schematic representation of the hydrolytic action of arabinoxylan degrading enzymes.

Product Code   Product Name                                                                                                      
endo-1,4-β-Xylanases                                                              
E-XYTR1 endo-1,4-β-Xylanase M1 (Trichoderma viride
E-XYTR3
endo-1,4-β-Xylanase M3 (Trichoderma longibrachiatum)                
E-XYAN4 endo-1,4-β-Xylanase M4 (Aspergillus niger
E-XYRU6 endo-1,4-β-Xylanase M6 (rumen microorganism)
E-XYNAP Recendo-1,4-β-Xylanase (Aeromonas punctata)
E-XYNBS Recendo-1,4-β-Xylanase (Bacillus stearothermophilus T6)
E-XYNACJ Recendo-1,4-β-Xylanase (Cellvibrio japonicus)
E-XYNBCM
Recendo-1,4-β-Xylanase (Cellvibrio mixtus)
E-XYLNP Recendo-1,4-β-Xylanase (Neocallimastix patriciarum)
E-XYLATM
Recendo-1,4-β-Xylanase (Thermotoga maritima)
exo-1,4-β-Xylosidases
E-BXSEBP  Recβ-D-Xylosidase (Bacillus pumilus)
E-BXSR
Recβ-D-Xylosidase (Selenomonas ruminantium)
α-L-Arabinofuranosidases
E-AFASE  α-L-Arabinofuranosidase (Aspergillus niger)
E-ABFAN  α-L-Arabinofuranosidase (Aspergillus nidulans
E-AFAM2   Recα-L-Arabinofuranosidase (Bifidobacterium adolescentis)
E-ABFCJ
Recα-L-Arabinofuranosidase (Cellvibrio japonicus)
E-ABFCT Recα-L-Arabinofuranosidase (Cellvibrio thermocellum
E-ABFUM
Recα-L-Arabinofuranosidase (Ustilago maydis)NEW
E-ABFBO17  Recα-L-Arabinofuranosidase (BACOVA 03417) (Bacteroides ovatus)NEW
E-ABFBO21  Recα-L-Arabinofuranosidase (BACOVA 03421) (Bacteroides ovatus)NEW
E-ABFBO25
Recα-L-Arabinofuranosidase (BACOVA 03425) (Bacteroides ovatus)NEW
Feruloyl Esterases
E-FAEZCT   RecFeruloyl Esterase (Clostridium thermocellum)
E-FAERU   RecFeruloyl Esterase (rumen microorganism)
Rec (recombinant enzyme)                                                                                                                              

Enzyme Substrates

Colourimetric oligosaccharides with a chemically-defined structure are available for the measurement of the activity of endo-xylanases, exo-xylosidases and α-L-arabinofuranosidases. These are suitable for use in both manual and high throughput automated analysis applications. The activity of the enzyme assayed can be determined directly by measuring the absorption increase at 400 nm which corresponds to the release of the nitrophenolate ion in solution. 

Product Code   Product Name                     Chemical Structure                                                     
endo-1,4-β-Xylanase substrates 
O-PNPX2  4-Nitrophenyl-β-
xylobioside 
O-PNPX2+3
 
O-PNPX3  4-Nitrophenyl-β-
xylotrioside
NEW        
exo-1,4-β-Xylanase substrates  
O-4MUX2 4-Methylumbelliferyl-β-
xylobioside
 
 4-Methylumbelliferyl-β-xylobioside
O-4MUX3
4-Methylumbelliferyl-β-
xylobiosideNEW 
O-PNPX  4-Nitrophenyl-β-D-
xylopyranoside
NEW 
O-PNPX
α-L-Arabinofuranosidase substrates 
O-PNPAF  4-Nitrophenyl-α-
L-arabinofuranoside
NEW
O-PNPAF
 


Insoluble and soluble chromogenic enzyme substrates 
derived from arabinoxylan and xylan (birchwood) have been prepared for the detection and specific measurement of endo-xylanase activity. These substrates produce a colourimetric response upon incubation with a suitable xylanase that can be related to enzyme activity through the standard curve provided (soluble substrates only) or used for screening on agar plates. The insoluble substrate is also available in tablet form to increase accuracy and convenience for the end-user for quantitative assays.

Soluble substrate overview
Figure: Schematic representation of the use of dyed polysaccharides (soluble) for the assay of endo-hydrolase activity.

Product Code  Product Name                                                                                                                                   
Tablets
T-XAX Xylazyme AX (very versatile, highly sensitive and widely used substrates for the assay of endo-xylanase)
T-XAF  Xylazyme AF (very versatile, highly sensitive and widely used substrates for the assay of endo-xylanase)
T-XYZ  Xylazyme (very versatile, highly sensitive and widely used substrates for the assay of endo-xylanase)
Soluble Substrates
S-AWAXL       Azo-Wheat Arabinoxylan (liquid)  
S-AWAXP Azo-Wheat Arabinoxylan (powder) 
S-AXBL  Azo-Xylan (birchwood) (liquid)
S-AXBP  Azo-Xylan (birchwood) (powder)
Insoluble Substrates
I-AZWAX  AZCL-Arabinoxylan (wheat) (useful for screening of endo-xylanase in gels and on agar plates)
I-AZXBW  AZCL-Xylan (birchwood) (useful for screening of endo-xylanase in gels and on agar plates)

1 McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by b-xylanase, α-L-arabinofuranosidase and b-xylosidase. Carbohydrate Research, 407, 75-96.