Providing an analytical solution

Enzyme Substrates

Discover our unrivalled range of exclusive oligosaccharides and for specific measurement of activity, suitable for analytical use and research.

Think Quality - Expect Excellence - Choose Megazyme Contents 1. Dyed Polysaccharides

The importance of in industrial processes and scientific research is constantly growing and the measurement of enzyme activity is of paramount importance for the use and characterisation of enzyme preparations.

Megazyme has unrivalled expertise in the production of pure enzymes and of substrates for the measurement of enzymatic activity. Our wide range of diverse enzymatic substrates that can be classified in three categories, as follows.

1. Dyed Polysaccharides a) Soluble substrates b) Insoluble substrates Suitable for gel/agar plate screening. Suitable for semi-quantitative assays. c) Tablet substrates Insoluble substrate formulated in tablets for quantitative assays and end-user convenience

 Suitable for the specific measurement ofendo -acting carbohydrate  Suitable for the analysis of crude enzymatic extracts or industrial preparations  Enzymatic activity determined by conversion from absorbance using a standard curve provided Dyed Polysaccharides Dyed  Generally not suitable for automated analytical assays (filtration step required)

2. Colourimetric Oligosaccharides a) Blocked oligosaccharides

For the analysis of crude enzymatic extracts or industrial preparations b) Non-blocked oligosaccharides Suitable only for the analysis of purified enzymes c) Colourimetric monosaccharides

 Suitable for the measurement of either endo-acting or exo-acting carbohydrate hydrolases

Colourimetric  Chemically defined substrates: no variability between substrate batches Oligosaccharides  Suitable for automated analytical assays and ideal for high-throughput applications  Enzymatic activity calculated directly from absorbance obtained  Fluorometric substrates also available for a higher sensitivity (fluorimeter required)

3. Carbohydrates a) Polysaccharides b) Oligosaccharides

 Wide range of highly purified polysaccharides and oligosaccharides available  Suitable for the measurement of either endo-acting or exo-acting carbohydrate hydrolases  Enzymatic activity determined by quantification of the increase in reducing sugar or decrease in viscosity of

Carbohydrates substrate solution  Polysaccharides: Native substrates of the enzymes being analysed  Oligosaccharides: Chemically defined substrates, no variability between substrate batches

2 Contents 1. Dyed Polysaccharides

Introduction to Dyed Polysaccharides

Dyed polysaccharides are useful substrates for the specific measurement of endo-acting hydrolases activity in crude plant extracts or industrial enzyme preparations.

In dyed polysaccharides, dye molecules are covalently attached to a or a partially depolymerised polysaccharide, to obtain substrates that are supplied as a Dyed Polysaccharides fine powder.

The endo-acting enzymes that recognise and act on these substrates release dyed fragments to an extent which is proportional to their enzymatic activity and which can be calculated by converting the absorbance obtained using a standard curve provided.

Dyed polysaccharide substrates offer the advantages of being specific and sensitive, and can form the basis of accurate, reliable and robust assay procedures, although they are not readily suited to automated assays due to a filtration or centrifugation step included in the assay protocol.

a) Soluble dyed substrates

Key features of Megazyme’s soluble dyed substrates

 Suitable for analysis of crude enzyme extracts  Suitable for analysis of purified enzymes  Quantitative assay  Chemically defined substrate  Suitable for gel/agar plate activity screening  Suitable for automation  Suitable for endo-acting enzymes  Suitable for exo-acting enzymes

3 Shop now at www.megazyme.com a) Soluble Dyed Substrates b) Insoluble Dyed Substrates

Assay procedure for soluble dyed substrates

The use of agar plates 1. The substrate is water soluble and is readily recognised and hydrolysed containing 0.5% w/v xanthan by an endo-acting hydrolase. gum avoids settling of the 2. The reaction is terminated by the addition of a precipitant solution (e.g. insoluble dyed substrates ethanol) wherein high molecular weight, partially hydrolysed substrate fragments precipitate from solution. 3. The suspension is mixed thoroughly, centrifuged, and the colour in the solution is measured using a spectrophotometer. 4. The enzymatic activity is determined using a standard curve provided.

Our range of soluble dyed substrates

Product Name Product Code Enzyme Measured Dyed Polysaccharides Dyed Red Starch S-RSTAR α-

Red Debranched Arabinan (sugar beet) S-RDAR endo-1,5-α-Arabinanase

Azo-CM-Cellulose (liquid) S-ACMCL

Azo-CM-Cellulose (powder) S-ACMC endo- / endo-1,4-β-Glucanase

Azo-Xyloglucan (tamarind) S-AZXG

Azo-Barley Glucan S-ABG100 / endo-b-Glucanase

Azo-Fructan S-AZFR5 endo-Fructanase / endo- Azo-Fructan plus exo-Inulinase S-AZFRXOI

Red S-RPUL / Limit-dextrinase

Azo-Carob Galactomannan S-ACGLM endo-1,4-β-Mannanase

Azo-Galactan (potato) S-AGALP endo-1,4-b-Galactanase

Azo-Casein (sulphanilamide dyed) S-AZCAS endo-Protease

AZ-Rhamnogalacturonan S-AZRH endo-Rhamnogalacturonan hydrolase /

Azo-Wheat Arabinoxylan (liquid) S-AWAXL

Azo-Wheat Arabinoxylan (powder) S-AWAXP endo-1,4-b- Azo-Xylan (birchwood) (liquid) S-AXBL

Azo-Xylan (birchwood) (powder) S-AXBP

4 a) Soluble Dyed Substrates b) Insoluble Dyed Substrates

Megazyme’s insoluble dyed substrates are prepared by dyeing and crosslinking soluble polysaccharides.

These substrates are provided in both granular and fine particle size. The finely milled substrates are more useful in agar plate assays and semi quantitative tube and plate assays. Substrates in other colours are also being developed for gel/agar plate activity screenings.

Insoluble chromogenic substrates are recommended for semi-quantitative assays. Dyed Polysaccharides

Key features of Megazyme’s insoluble dyed substrates

 Suitable for analysis of crude enzyme extracts  Quantitative assay  Suitable for analysis of purified enzymes  Chemically defined substrate  Suitable for gel/agar plate activity screening  Suitable for automation  Suitable for endo-acting enzymes  Suitable for exo-acting enzymes

Assay procedure for insoluble dyed substrates The use of insoluble dyed substrates in agar plates containing 0.5% w/v xanthan 1. The hydrated insoluble substrate forms gelatinous particles which are readily gum allows enzymatic activity recognised and hydrolysed by an endo-acting hydrolase. The partial detection more rapidly and at of insoluble chromogenic substrates releases water soluble dyed substrate lower enzyme concentration fragments. 2. The reaction is terminated by adding an alkaline solution to stop enzyme activity and the reaction slurry is filtered or centrifuged. 3. The colour released in solution is measured using a spectrophotometer and the colour intensity is directly related to enzyme activity.

5 Shop now at www.megazyme.com b) Insoluble Dyed Substrates c) Tablet Substrates

Our range of insoluble dyed substrates Product Name Product Code Enzyme Measured AZCL-Amylose I-AZAMY AZCL-Amylose (fine) I-AZAMYF a-Amylase RedCL-Amylose (fine) I-RCLAMYF AZCL-Arabinan (debranched) I-AZDAR endo-1,5-α-Arabinanase AZCL-HE-Cellulose I-AZCEL AZCL-HE-Cellulose (fine) I-AZCELF RedCL-HE-Cellulose (fine) I-RCLCELF Azo-α-Cellulose I-ACELL endo-Cellulase Azo-Avicel I-AAVIC AZCL-Xyloglucan I-AZXYG AZCL-Xyloglucan (fine) I-AZXYGF RedCL-Xyloglucan (fine) I-RCLXYGF AZCL-Chitosan I-AZCHAN endo- Dyed Polysaccharides Dyed AZCL-Chitosan (fine) I-AZCHANF AZCL-Dextran I-AZDEX endo-1,6-α-Dextrinase AZCL-Dextran (fine) I-AZDEXF AZCL-Galactan (potato) I-AZGLP endo-1,4-b-Galactanase AZCL-Galactan (potato) (fine) I-AZGLPF AZCL-Curdlan I-AZCUR AZCL-Curdlan (fine) I-AZCURF endo-1,3-b-Glucanase RedCL-Curdlan (fine) I-RCLCURF AZCL-Pachyman I-AZPAC AZCL-Barley b-Glucan I-AZBGL AZCL-Barley b-Glucan (fine) I-AZBGLF Lichenase / endo-b-Glucanase RedCL-Barley b-Glucan (fine) I-RCLBGLF AZCL-Galactomannan I-AZGMA AZCL-Galactomannan (fine) I-AZGMAF endo-1,4-b-Mannanase RedCL-Galactomannan (fine) I-RCLGMAF AZCL-Casein I-AZCAS endo-Protease AZCL-Collagen I-AZCOL AZCL-Pullulan I-AZPUL AZCL-Pullulan (fine) I-AZPULF Pullulanase / Limit-Dextrinase RedCL-Pullulan (fine) I-RCLPULF AZCL-Rhamnogalacturonan 1 I-AZRHI Rhamnogalacturonan hydrolase / lyase AZCL-Arabinoxylan (Wheat) I-AZWAX AZCL-Arabinoxylan (Wheat) (fine) I-AZWAXF endo-1,4-b-D-Xylanase AZCL-Xylan (Birchwood) I-AZXBW RedCL-Arabinoxylan (Wheat) (fine) I-RCLWAXF

6 b) Insoluble Dyed Substrates c) Tablet Substrates

Megazyme supplies a range of enzyme tablet tests for ultimate end-user convenience. The insoluble chromogenic substrates discussed above are formulated into tablets. By using tablet tests, it is possible to obtain quantitative determinations of endo-acting hydrolase activities.

From a procedural perspective, tablet tests operate in the same way as assays using insoluble chromogenic substrates. The advantage of enzyme tablet tests is that the need to accurately weigh substrate quantities (and the error associated with this parameter) is removed.

Key features of Megazyme’s tablet substrates Dyed Polysaccharides

 Suitable for analysis of crude enzyme extracts  Chemically defined substrate  Suitable for analysis of purified enzymes  Suitable for automation  Quantitative assay  Suitable for gel/agar plate activity screening  Suitable for endo-acting enzymes  Suitable for exo-acting enzymes

Our range of tablet substrates

Product Name Product Code Enzyme Measured Amylazyme T-AMZ Amylazyme BG T-AMZBG α-Amylase Amylazyme Red T-AMZRD Amylazyme HY T-AMZHY Arabinazyme T-ARZ endo-1,5-α-Arabinanase Cellazyme AF (40mg) T-CAF Cellazyme C (60mg) T-CCZ endo-Cellulase / endo-1,4-b-Glucanase Cellazyme T T-CTZ Chitozyme T-CHZ endo-Chitosanase α-Dextrazyme T-DEXT endo-1,6-α- b-Glucazyme T-BGZ Lichenase / endo-b-Glucanase Galactazyme T-GLZ endo-1,4-b-Galactanase 1,3-b-Glucazyme T-PAZ endo-1,3-b-Glucanase 1,3-b-Glucazyme HS T-CUR Limit-Dextrizyme T-LDZ Pullulanase / Limit-dextrinase Mannazyme T-MNZ endo-1,4-b-Mannanase Protazyme AK T-PRAK endo-Protease Protazyme OL T-PROL Rhamnozyme T-RHAM Rhamnogalacturonan hydrolase / lyase Psyllazyme T-PSYL Xylazyme AF (40mg) T-XAF endo-1,4-b-Xylanase Xylazyme AX (60mg) T-XAX Xylazyme (100mg) T-XYZ

7 Shop now at www.megazyme.com 2. Colourimetric Oligosaccharides a) Blocked Oligosaccharides

Introduction to Colourimetric Oligosaccharides

Colourimetric substrates are useful for the specific measurement of endo-acting and exo-acting carbohydrate hydrolase activity. The molecular structure of these substrates is chemically-defined which completely removes the possibility of any ‘batch to batch’ variability.

These substrates are prepared by installing a covalently bound colourimetric or fluorometric moiety onto the reducing end sugar residue of an oligosaccharide and are usually supplied as a fine powder or as a solution. The endo- or exo-acting hydrolase enzymes that recognise and act on these substrates release the colourimetric or fluorometric moiety to an extent which is proportional to their enzymatic activity and which can easily be calculated using the applicable extinction coefficient.

Colourimetric oligosaccharides offer the advantages of being specific and sensitive, and can be used in fully automated assay formats owing to the fact that there is no filtration step required in these assays. Assay formats based on these substrates are the most convenient for the user and display the highest levels of reproducibility.

a) Blocked Oligosaccharides

Blocked colourimetric substrates for the measurement of carbohydrate hydrolase activity are exclusively offered by Megazyme and are an excellent alternative to dyed polysaccharides for the quantitative measurement of endo- acting hydrolase activity in crude enzyme preparations.

No variability between substrate batches occurs due to the chemically-defined molecular structures of these

substances. These substrates are usually available as components within assay kits and are used in enzyme- coupled assay protocols.

The blocking group at the non-reducing end of the substrate prevents the activity of exo-acting enzymes present in a crude mixture and their structure makes them absolutely specific targets for the endo-acting enzyme being analysed. Colourimetric Oligosaccharides

Key features of Megazyme’s blocked oligosaccharides

 Suitable for analysis of crude enzyme extracts  Suitable for analysis of purified enzymes  Chemically defined substrate  Quantitative assay  Suitable for automation  Suitable for gel/agar plate activity screening  Suitable for endo-acting enzymes  Suitable for exo-acting enzymes

8 2. Colourimetric Oligosaccharides a) Blocked Oligosaccharides

Assay procedure for blocked oligosaccharides

1. The soluble blocked colourimetric substrate is readily recognised and hydrolysed by an endo-acting hydrolase. The hydrolysis of the substrate releases two components in solution and the ancillary exo-acting enzyme provided is then able to further hydrolyse the colourimetric group bearing fragment to release the colourimetric group in solution (e.g. 4-nitrophenol). 2. The reaction is terminated by adding an alkaline solution to stop enzyme activity. 3. The colour release in solution is measured using a spectrophotometer and the colour intensity is directly related to enzyme activity.

Our range of blocked oligosaccharides

Product Product Name Enzyme Measured Code α-Amylase Assay Kit (Ceralpha method) K-CERA α-Amylase α-Amylase SD Assay Kit (High sensitivity method) K-AMYLSD α-Amylase Reagent (Ceralpha) R-CAAR4 Amylase HR Reagent R-AMHR4 Blocked 4-nitrophenyl-α-maltoheptaoside O-BPNPC7 Cellulase Assay Kit (CellG5 method) K-CellG5 Cellulase / endo-1,4-b-Glucanase

Cellulase Assay Kit (CellG3 method) K-CellG3 Oligosaccharides Colourimetric Blocked 4-methylumbelliferyl-b-cellotrioside R-CELLFLR (Cellafluor reagent) Blocked 4-methylumbelliferyl-b-cellotrioside O-B4MUG3 Blocked b-naphthyl-b-cellotrioside O-BNAPG3 Lichenase / endo-b-Glucanase

Malt b-Glucanase / Lichenase Assay Kit K-MBG4 (MBG4 method)

Pullanase / Limit-Dextrinase

Pullulanase / Limit-Dextrinase Assay Kit K-PullG6 (PullG6 method)

endo-1,4-b-Xylanase

endo-Xylanase Assay Kit K-XylX6 (XylX6 method)

9 Shop now at www.megazyme.com b) Non-Blocked Oligosaccharides b) Non-Blocked Oligosaccharides

Non-blocked colourimetric substrates are generally used for the quantitative measurement of endo-acting hydrolase activity in purified enzyme preparations. The lack of a blocking-group makes these substrates unsuitable for the analysis of crude enzymatic mixtures because of the likely presence of competing exo-acting enzymes. In non-blocked oligosaccharide substrates, an oligosaccharide chain of defined length is covalently linked to either a colourimetric (4-nitrophenol or 2-chloro-4-nitrophenol) or a fluorometric (4-methylumbelliferone) group.

Purified endo-acting enzymes have very specific requirements. The possibility of screening substrates having different chain length allows for a sensitive and specific characterisation of endo-acting carbohydrate hydrolases. Researchers can derive valuable information from the increase in absorbance at 400 nm (or lack thereof) or indeed by HPLC analysis of enzyme incubations with colourimetric oligosaccharides of varying length.

The use of fluorometric substrates is similar to that of colourimetric ones from a procedural perspective. They generally display a higher sensitivity, however a fluorimeter is required to measure the release of the fluorometric group in solution.

Key features of Megazyme’s non-blocked oligosaccharides

Colourimetric  Suitable for analysis of purified enzymes  Suitable for analysis of crude enzyme extracts Oligosaccharides  Chemically defined substrate  Quantitative assay  Suitable for automation  Suitable for gel/agar plate activity screening  Suitable for endo-acting enzymes  Suitable for exo-acting enzymes

Our range of non-blocked oligosaccharides

Product Product Name Enzyme Measured Code 2-Chloro-4-nitrophenyl-β-(1,3:1,4)-glucotrioside DP3 O-CNPBG3 Substrates for Lichenase / endo-(1,3:1,4)-b-Glucanase 2-Chloro-4-nitrophenyl-β-(1,3:1,4)-glucotetraoside DP4 O-CNPBG4 4-Methylumbelliferyl-β-(1,3:1,4)-glucotrioside DP3 O-4MUBG3

10 b) Non-Blocked Oligosaccharides b) Non-Blocked Oligosaccharides

Product Product Name Enzyme Measured Code Substrates for Cellulase / endo-1,4-b-Glucanase 2-Chloro-4-Nitrophenyl-β-Cello-Oligosaccharides: 2-Chloro-4-nitrophenyl-β-cellobioside DP2 O-CPNPG2 2-Chloro-4-nitrophenyl-β-cellotrioside DP3 O-CPNPG3 2-Chloro-4-nitrophenyl-β-cellotetraoside DP4 O-CPNPG4 2-Chloro-4-nitrophenyl-β-cellopentaoside DP5 O-CPNPG5 4-Methylumbelliferyl-β-Cello-Oligosaccharides: 4-Methylumbelliferyl-β-cellobioside DP2 O-4MUG2 4-Methylumbelliferyl-β-cellotrioside DP3 O-4MUG3 4-Methylumbelliferyl-β-cellotetraoside DP4 O-4MUG4 4-Methylumbelliferyl-β-cellopentaoside DP5 O-4MUG5 Substrates for endo-1,3-b-Glucanase 4-Nitrophenyl-β-Laminari-Oligosaccharides: 4-Nitrophenyl-β-laminaribioside DP2 O-PNPLAM2 4-Nitrophenyl-β-laminaritrioside DP3 O-PNPLAM3 4-Nitrophenyl-β-laminaritetraoside DP4 O-PNPLAM4 4-Methylumbelliferyl-β-Laminari-Oligosaccharides:

4-Methylumbelliferyl-β-laminaribioside DP2 O-4MULAM2 Oligosaccharides Colourimetric 4-Methylumbelliferyl-β-laminaritrioside DP3 O-4MULAM3 4-Methylumbelliferyl-β-laminaritetraoside DP4 O-4MULAM4 Substrates for endo-1,4-b-Mannanase 2-Chloro-4-Nitrophenyl-a-Manno-Oligosaccharides: 2-Chloro-4-nitrophenyl-a-mannobioside DP2 O-CPNPAM2

2-Chloro-4-nitrophenyl-a-mannotrioside DP3 O-CPNPAM3 2-Chloro-4-nitrophenyl-a-mannotetraoside DP4 O-CPNPAM4 4-Methylumbelliferyl-a-Manno-Oligosaccharides: 4-Methylumbelliferyl-a-mannobioside DP2 O-4MUAM2 4-Methylumbelliferyl-a-mannotrioside DP3 O-4MUAM3 4-Methylumbelliferyl-a-mannotetraoside DP4 O-4MUAM4 Substrates for endo-1,4-b-Xylanase 4-Nitrophenyl-β-Xylo-Oligosaccharides: 4-Nitrophenyl-β-xylobioside DP2 O-PNPX2 4-Nitrophenyl-β-xylotrioside DP3 O-PNPX3 2-Nitrophenyl-β-xylobioside DP2 O-ONPX2 4-Methylumbelliferyl-β-Xylo-Oligosaccharides: 4-Methylumbelliferyl-β-xylobioside DP2 O-4MUX2 4-Methylumbelliferyl-β-xylotrioside DP3 O-4MUX3

11 Shop now at www.megazyme.com c) Colourimetric substrates for c) Colourimetric substrates for exo-acting carbohydrate hydrolases exo-acting carbohydrate hydrolases

Colourimetric monosaccharides are commonly used for the quantitative measurement of exo-acting hydrolase activity in either purified or crude enzyme preparations. Colourimetric monosaccharide substrates contain a colourimetric group (4-nitrophenol) either α- or β-linked to a monosaccharide.

Key features of Megazyme’s colourimetric substrates for exo-acting carbohydrate hydrolases

 Suitable for analysis of purified enzymes  Suitable for analysis of crude enzyme extracts  Chemically defined substrate  Quantitative assay

 Suitable for automation  Suitable for gel/agar plate activity screening  Suitable for exo-acting enzymes  Suitable for endo-acting enzymes

Assay procedure for colourimetric substrates for exo-acting carbohydrate hydrolases

Colourimetric 1. Once a soluble colourimetric monosaccharide substrate is hydrolysed by an exo-acting hydrolase, the Oligosaccharides colourimetric group is released in solution. 2. The reaction is terminated by adding an alkaline solution to stop enzyme activity. 3. The colour release in solution is measured using a UV spectrophotometer and the colour intensity is directly related to enzyme activity.

12 c) Colourimetric substrates for c) Colourimetric substrates for exo-acting carbohydrate hydrolases exo-acting carbohydrate hydrolases

Our range of colourimetric substrates for exo-acting carbohydrate hydrolases

Product Name Product Code Enzyme Measured

β-Amylase Assay Kit (Betamyl-3) K-BETA3 4-Nitrophenyl-β-maltotrioside + β-glucosidase β-Amylase Assay Reagent (Betamyl-3) R-BAMR3 4-Nitrophenyl-β-maltotrioside + β-glucosidase b-Amylase 4-Nitrophenyl-β-maltotrioside O-PNPC3

b-Naphthyl-β-maltotrioside O-NAPC3 4-Nitrophenyl-β-maltoside + β-glucosidase R-AMGR3 Amyloglucosidase (Amyloglucosidase Assay Reagent) 4-Methylumbelliferyl-β-N-acetyl-D-glucosaminide O-4MUNAG β-N-Acetyl-D-Glucosaminidase 4-Nitrophenyl-β-N-acetyl-D-glucosaminide O-PNPNAG

4-Nitrophenyl-α-L-arabinofuranoside O-PNPAF α-L-Arabinofuranosidase

4-Nitrophenyl-α-D-glucopyranoside O-PNPAG α-Glucosidase

4-Nitrophenyl-b-D-glucopyranoside O-PNPBG b-Glucosidase

4-Nitrophenyl-α-D-galactopyranoside O-PNPAGAL α-Galactosidase Oligosaccharides Colourimetric

4-Nitrophenyl-b-D-galactopyranoside O-PNPBGAL β-Galactosidase

4-Nitrophenyl-α-D-glucuronide O-PNPAGA α-Glucuronidase

4-Nitrophenyl-β-D-glucuronide O-PNPBGA β-Glucuronidase

4-Nitrophenyl-α-Nitrophenyl-α-D-galacturonide O-PNPAGALA α-Galacturonidase

4-Nitrophenyl-b-Nitrophenyl-α-D-galacturonide O-PNPBGALA β-Galacturonidase

4-Nitrophenyl-α-L-fucopyranoside O-PNPAFC α-

4-Nitrophenyl-α-D-mannopyranoside O-PNPAM α-

4-Nitrophenyl-β-D-mannopyranoside O-PNPBM β-Mannosidase

4-Nitrophenyl-α-D-xylopyranoside O-PNPAX α-Xylosidase

4-Nitrophenyl-β-D-xylopyranoside O-PNPX β-Xylosidase

4-Nitrophenyl-β-lactoside O-PNPL Cellobiohydrolase

4-Nitrophenyl-N,N’-diacetyl-β-chitobioside O-PNPCH2 Chitobiosidase 4-Methylumbelliteryl-N,N’-diacetyl-β-chitobioside O-4MUCH2

13 Shop now at www.megazyme.com Carbohydrates nye xrcs ht a aray oti rdcn sgr.Vsoiy euto mtos r seii for specific are methods reduction Viscosity sugars. reducing contain already may that extracts enzyme Reducing-sugar methods can be used for the assay of pure enzyme solutions but are unsuitable for use with crude substrate solutiononhydrolysis.the of viscosity in decrease or sugar reducing in increase the of quantification byfollowed substrate, enzymatic Traditionally, a) Polysaccharides site requirements, bindingaffinities andactivities. substrates native the as oligosaccharides, and encountered by polysaccharides carbohydrate hydrolases enzymes, in nature, hydrolyticcan give the oftruest insight into assaytheir mechanism of the action, active convenient for most methods the certainly are assays oligosaccharide-based colourimetric and polysaccharide dyed While foremost research institutes inthe world. Our global supplier Megazyme offers abroad range of carbohydrates for scientists involved inenzymeresearch, actingasthe Introduction to Carbohydrates hydrolase activity butare tedious to perform andrequire specialist equipment.     Key features of Megazyme’s polysaccharides

Suitable for analysis of purifiedenzymes best-in-class purity Suitable for Suitable for gel/agar plate activity screening Quantitative assay endo for anumber of uniquecarbohydrates. endo -acting carbohydrate hydrolases have been measured using the native polysaccharide as the as polysaccharide native the using measured been have hydrolasescarbohydrate -acting -acting enzymes andreputation for quality have resulted inlongstandingrelationships with someof the 3. Carbohydrates

O 14     Suitable for Suitable for automation Chemically defined substrate Suitable for analysis of crudeenzymeextracts O exo -acting enzymes sole endo - a) Polysaccharides Carbohydrates www.megazyme.com -Glucanase

b -1,4- -Glucanase b endo - Shop now at Shop now endo -Dextrinase -Arabinanase -Glucanase -Galactanase -Galactanase α b b b α - b -1,3- - -1,6- -Fructanase -1,3- -1,4- -1,5- -Cellulase / -Amylase endo endo endo endo endo endo Lichenase / Enzyme Activity Enzyme a endo endo 15 P-BGCFA P-CURDL P-GLCML P-GLCMH P-CHITIN P-DEXT P-FOS28 P-INUL P-LEVAN P-ARGAL P-GALLU P-GALPOT P-PGALU P-PGAPT P-LICHN P-MWBGS P-BGBL P-BGBM P-BGBH P-BGOM P-BGOH Cat. No. Cat. P-AMYL P-BLDX P-ARAB P-DBAR P-LARB P-CMLA P-CMC4M P-XYGLN P-CMCUR P-PACHY P-CMPAC P-BGYST a) Polysaccharides -L-Arabinan (sugar beet) (sugar -L-Arabinan α -L-Arabinan (sugar beet) (sugar -L-Arabinan α NEW

-Glucan (oat; medium viscosity) medium -Glucan (oat; viscosity) high -Glucan (oat; standard -Glucan CFA -Glucan (barley; medium viscosity) medium -Glucan (barley; viscosity) high -Glucan (barley; -Glucan MW standard standard -Glucan MW viscosity) low -Glucan (barley; -Limit -Glucan (yeast; alkali soluble) alkali -Glucan (yeast; Levan Curdlan b b b b b b b Pectic Galactan (potato) Pectic moss) Lichenan (Icelandic Galactan (lupin) Galactan (potato) Galactan (lupin) Pectic Arabinogalactan (larch wood) (larch Arabinogalactan Fructooligosaccharides (DP2-8) Fructooligosaccharides Inulin (DP2-60) Chitin (colloidal) Dextran Xyloglucan (tamarind) Xyloglucan viscosity) low (konjac; Glucomannan viscosity) high (konjac; Glucomannan CM-Linear 1,5- CM-Linear 4M Cellulose Carboxymethyl Debranched Arabinan (sugar beet) (sugar Arabinan Debranched 1,5- Linear b beet) (sugar Arabinan Product (potato) Amylose b Pachyman CM-Pachyman CM-Curdlan Our range of polysaccharides of range Our 3. Carbohydrates Carbohydrates Arabinoxylan (enzyme debranched; 30%arabinose) Arabinoxylan (aciddebranched; 26%arabinose) Arabinoxylan (aciddebranched; 22%arabinose) Arabinoxylan (wheat flour;insoluble) Arabinoxylan (wheat flour;high viscosity) Arabinoxylan (wheat flour;medium viscosity) Arabinoxylan (wheat flour;low viscosity) Arabinoxylan (rye flour) Xylan (beechwood; purified) Rhamnogalacturonan (soy bean) Rhamnogalacturonan I(potato) Polygalacturonic Acid (PGA) Pullulan (NaBH Pullulan (guar; galactose depleted; 28%galactose) Galactomannan (guar; galactose depleted; 21% galactose) Galactomannan Galactomannan (guar; high viscosity) Galactomannan (guar; medium viscosity) Galactomannan (carob; high viscosity) Galactomannan (carob; low viscosity) Mannan (1,4- Mannan (ivory nut) Product β -D-mannan) 4 reduced) a) Polysaccharides P-GGM28 P-GGM21 P-GGMHV P-GGMMV P-GALMH P-GALML P-MANCB P-MANIV P-EDWAX30 P-ADWAX26 P-ADWAX22 P-WAXYI P-WAXYH P-WAXYM P-WAXYL P-RAXY P-XYLNBE P-RHAGN P-RHAM1 P-PGACT P-PULLBH P-PULLN Cat. No. 16 endo endo Rhamnogalacturonan hydrolase /lyase endo Pullulanase /Limit-Dextrinase Enzyme Activity -Polygalacturonanase / Polygalacturonate lyase -1,4- -1,4- b β -Mannanase -Xylanase b) Oligosaccharides Carbohydrates www.megazyme.com

Shop now at Shop now -acting carbohydrate hydrolase enzymes, the exo Suitable for analysis of crude enzyme extracts of analysis Suitable for automation Suitable for screening activity plate gel/agar Suitable for - or 17    endo b) Oligosaccharides b) -acting enzymes -acting enzymes endo exo substrate defined Chemically Suitable for Suitable for Quantitative assay assay Quantitative Suitable for analysis of purified enzymes of analysis Suitable for

Oligosaccharides can also be employed as building blocks for the chemical synthesis of either colourimetric colourimetric either of synthesis chemical the for blocks building as employed be also can Oligosaccharides interest. enzymes of of inhibitors or substrates Our range of oligosaccharides of range Our      Key features of Megazyme’s oligosaccharides Megazyme’s of features Key Oligosaccharides Oligosaccharides can also be employed as building interest. enzymes of of blocks inhibitors or substrates for the chemical synthesis of either colourimetric A A number of borohydride reduced oligosaccharides are also These available. are particularly useful substrates in where the reducing sugar assays native oligosaccharides, being themselves reducing sugars, can produce a high assay. the of the sensitivity reducing thereby value, absorbance ‘blank’ molecules can be used to help analysis. HPLC/HPAEC-PAD characterise or methods sugar reducing through can be measured which either of activity Defined Defined oligosaccharide substrates are particularly useful for researchers in the field of glycoscience. These a) Polysaccharides b) Oligosaccharides b) Oligosaccharides

N-Acetyl-Chitooligosaccharides Substrates for endo-1,4-chitinase, chitobiosidase and N-acetyl-β-D-glucosaminidase. O-CHI2 DP2 Diacetyl-chitobiose O-CHIN2 DP2 1,4-b-D-Glucosaminyl-D-N-Acetylglucosamine (HCl) NEW O-CHI3 DP3 Triacetyl-chitotriose O-CHI4 DP4 Tetraacetyl-chitotetraose O-CHI5 DP5 Pentaacetyl-chitopentaose O-CHI6 DP6 Hexaacetyl-chitohexaose

Amylosaccharides (mixed-linkage) Substrates for limit-dextrinase, pullulanase, , , amyloglucosidase and α-glucosidase.

O-PAN Panose O-IPAN Isopanose O-GMT 63-α-D-Glucosyl-

3 O-GMH 63-α-D-Glucosyl-maltotriosyl-maltriose O-MTMT 6 -α-D-Maltotriosyl-maltotriose 3 O-MTMTRD 6 -α-D-Maltotriosyl-maltotriitol (BH4 reduced) 1,5-α-L-Arabinooligosaccharides Substrates for endo-1,5-α-arabinanase and α-arabinofuranosidase. O-ABI DP2 Arabinobiose (syrup) O-ATR DP3 Arabinotriose (syrup) O-ATE DP4 Arabinotetraose (syrup) O-APE DP5 Arabinopentaose (syrup) O-AHE DP6 Arabinohexaose (powder) O-AHP DP7 Arabinoheptaose (powder)

Carbohydrates O-AOC DP8 Arabino-octaose (powder)

O-A4B 32-α-L-Arabinofuranosyl-(1,5)-α-L-arabinotriose O-A5BMIX 22 32-di-α-L-Arabinofuranosyl-(1,5)-α-L-arabinotriose plus 32-α-L-arabinofuranosyl-(1,5)-α-L-arabinotetraose

18 Carbohydrates

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-D-Glucosyl-cellotriose β -D-Cellobiosyl-cellobiose -D-Cellobiosyl-cellobiose -D-Cellobiosyl- - β β 3 - - 1 2 3 Shop now at Shop now 3 + 3 O-BGTRIB O-BGTETC 19 -. -glucosidase and lipid polysaccharide monooxygenase (LPMO). endo β -glucosidase. β NEW NEW

-D-Cellotetraitol -D-Cellopentaitol -D-Cellohexaitol -D-Cellotriitol -D-Galactosyl-D-glucose) b b b b b -fructosidase and b b) Oligosaccharides b) Cellotriose Cellotetraose Cellopentaose Cellohexaose 1,4- 1,4- 1,4- 1,4- -glucanase and β 1,1-Kestotetraose 1,1,1-Kestopentaose Levanbiose Levantriose 1-Kestose Inulotriose

-D-Galactobiose -D-Galactobiose -D-Galactosyl-D-glucose -D-Galactobiose -D-Glucosyl-cellobiose -D-Cellotriosyl-glucose b b b b β -inulinase, -cellulase, cellobiohydrolase, β - - 1 2 DP3 DP4 DP5 DP6 3 3 DP4 DP5 DP6 DP3 DP4 DP5 DP2 DP3 DP3 DP3 1,6- 1,3- Allolactose (1,6- 1,3- 1,4- -galactosidase. endo endo b

-D-Glucooligosaccharides b O-GBI6 O-LAC3 O-LAC6 O-GBI3 O-GBI4 O-CPE O-CHE O-CTR O-CTE O-BGTETB O-BGTRIA O-CHERD O-CTRRD O-CTERD O-CPERD O-LEV2 O-LEV3 O-KTE O-KPE O-INU3 O-KTR Substrates for Substrates for Substrates for Substrates for lichenase/ Galactooligosaccharides (GOS) Galactooligosaccharides Fructooligosaccharides (FOS) Fructooligosaccharides 1,3:1,4- Cellooligosaccharides -L-arabinotetraose α -L-arabinofuranosyl-(1,5)- α - 2 -L-arabinotriose -L-arabinotriose 3 plus α -L-arabinotriose α b) Oligosaccharides b) -L-Arabinofuranosyl-(1,5)- α -di- -L-Arabinofuranosyl-(1,5)- 2 α 3 - 2 2 2 3 2

O-A4B O-A5BMIX b) Oligosaccharides b) Oligosaccharides

Galactomannooligosaccharides Substrates for endo-1,4-β-mannanase, β-mannosidase and α-galactosidase.

1 O-GM3 6 -α-D-Galactosyl-mannotriose O-GMM3 61-α-D-Galactosyl-mannobiose plus mannotriose

O-GGM5 63, 64-α-D-Galactosyl-mannopentaose

Galactosyl-Sucrose Oligosaccharides Substrate for α-galactosidase.

O-VER Verbascose

Glucomannooligosaccharides Substrates and standards for glucomannan degrading enzymes.

O-GM 1,4-β-D-Glucosyl-D-Mannose O-GGM 1,4-β-D-Cellobiosyl-D-Mannose O-GMM 1,4-β-D-Glucosyl-D-Mannobiose O-MGM 1,4-β-D-Mannosyl-1,4-β-D-Glucosyl-D-Mannose

O-GMMBI 1,4-β-D-Glucosyl-D-Mannobiose plus 1,4-β-D-Cellobiosyl-D-Mannose

1,3-b-Glucooligosaccharides Substrates for endo-1,3-β-glucanase (laminarinase), exo-1,3-β-glucanase and β-glucosidase O-LAM2 DP2 Laminaribiose

Carbohydrates O-LAM3 DP3 Laminaritriose O-LAM4 DP4 Laminaritetraose O-LAM5 DP5 Laminaripentaose O-LAM6 DP6 Laminarihexaose

20 b) Oligosaccharides b) Oligosaccharides

Gluco-diglucodisaccharides (various linkages) Substrates for a-glucosidase and b-glucosidase. O-GENT Glcβ1,6GIc Gentiobiose O-LAM2 Glcb1,3Glc Laminaribiose O-SOPH Glcb1,2Glc Sophorose O-IMO2 Glca1,6Glc Isomaltose O-NGR Glca1,3Glc Nigerose NEW O-KOJ Glca1,2Glc Kojibiose NEW Isomaltooligosaccharides Substrates for endo-1,6-a-dextranase and oligo-a-1,6-glucosidase. O-IMO2 DP2 Isomaltose O-IMO3 DP3 Isomaltotriose Maltooligosaccharides Substrates for a-amylase and b-amylase. O-MAL3 DP3 Maltotriose O-MAL5 DP5 Maltopentaose NEW O-MAL3RD DP3 Maltotriitol O-MAL6 DP6 Maltohexaose NEW O-MAL4 DP4 Maltotetraose O-MAL7 DP7 Maltoheptaose NEW O-MAL8 DP8 Maltooctaose NEW

1,4-b-D-Mannooligosaccharides Substrates for endo-1,4-β-mannanase and β-mannosidase. O-MBI DP2 Mannobiose O-MTR DP3 Mannotriose O-MTE DP4 Mannotetraose O-MPE DP5 Mannopentaose O-MHE DP6 Mannohexaose Xyloglucan Oligosaccharides Substrates for α-xylosidase and endo-1,4-β-glucanase.

O-X3G4 Xyloglucan heptasaccharide (X3Glc4)

O-X3G4R Heptasaccharide (X3Glc4; borohydride reduced) O-XGHON Xyloglucan (Hepta + octa + nona saccharides) O-XGHDP Higher DP xyloglucan oligosaccharides Carbohydrates O-IPRM Isoprimeverose (xyloglucan derived)

O-XCBIR Xylosyl-cellobiose (borohydride reduced)

21 Shop now at www.megazyme.com b) Oligosaccharides b) Oligosaccharides

1,4-b-D-Xylooligosaccharides Substrates for endo-1,4-β-xylanase and β-xylosidase. O-XBI DP2 Xylobiose O-XTR DP3 Xylotriose O-XTE DP4 Xylotetraose O-XPE DP5 Xylopentaose O-XHE DP6 Xylohexaose O-XBIRD DP2 1,4-β-D-Xylobiitol O-XTRRD DP3 1,4-β-D-Xylotriitol

Arabinoxylooligosaccharides Substrates for α-arabinofuranosidase, b-xylosidase and endo-1,4-β-xylanase

O-A3X 32-α-L-Arabinofuranosyl-xylobiose (A3X)

O-A2XX 23-α-L-Arabinofuranosyl-xylotriose (A2XX)

O-XA3XX 33-α-L-Arabinofuranosyl-xylotetraose (XA3XX)

O-A23XX 23-,33-di-α-L-Arabinofuranosyl-xylotriose (A2+3XX)

O-XAXXMIX 33-α-L- plus 23-α-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) mixture Carbohydrates

22 b) Oligosaccharides b) Oligosaccharides

Glucuronoxylooligosaccharides Substrates for endo-1,4-β-xylanase and a-glucuronidase.

O-UX 22-(4-O-Methyl-α-D-Glucuronyl)-xylobiose NEW

O-UXX 23-(4-O-Methyl-α-D-Glucuronyl)-xylotriose NEW O-UXXR Aldotetrauronic acid (borohydride reduced)

O-XUX 22-(4-O-Methyl-α-D-Glucuronyl)-xylotriose NEW

O-XUXX 23-(4-O-Methyl-α-D-Glucuronyl)-xylotetraose NEW O-XUXXR Aldopentaaouronic acid (borohydride reduced)

O-AMX Aldouronic acids mixture O-AMXR Aldouronic acids mixture (NaBH4 reduced)

Oligogalacturonides Substrates for a-galacturonidase.

O-GALA2 Digalacturonic acid Carbohydrates

23 Shop now at www.megazyme.com t + 353 1 286 1220 (worldwide) t + 1 312 212 4361 (USA) e [email protected]

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03/2019