tion www.megazyme.com risa acte har C nd a ay ss A e m y z n E
e s a r
d
y
h
o
b
r
a
C
r
o
f
s
n
o
i
t
u
l
o
S
e
t
e
l
p
m
o
C
-
s
e
t
a
r
t
s
b
u
S
e
m y z n E
Enzyme Substrates Complete Solutions for Carbohydrase Enzyme Assay and Characterisation ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Oligosaccharide ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Carbohydrate Substrates Carbohydrate Polysaccharide ✓ ✓ ✓ ✓ ✓ ✓ Non-blocked colourimetric ✓ ✓ ✓ ✓ ✓ Blocked Oligosaccharide Substrates Oligosaccharide ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Tablet ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Insoluble ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Soluble Dyed Polysaccharide Substrates Polysaccharide Dyed -Acting Carbohydrate Hydrolase Substrates Hydrolase -Acting Carbohydrate endo -Chitinase β -Glucanase -Inulinase β -Glucanase β -1,4- endo -Xylanase endo -Glucanase -1,4- -Mannanase β -Dextranase -Galactanase -Arabinanase β β α β -1,3:1,4- -Protease α -Amylase -Amylase endo α -1,4- endo -1,3- endo -1,4- -1,6- -1,4- -1,5- Enzyme Activity Enzyme endo endo endo endo endo endo -Fructanase / Pullulanase / Limit-dextrinase endo -Chitosanase / Cellulase / Lichenase / Rhamnogalacturonan hydrolase / lyase hydrolase Rhamnogalacturonan endo
1 Enzyme Substrates The importance of enzymes 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. It offers a wide range of diverse enzymatic substrates that can be classified in three categories: dyed polysaccharides, colourimetric oligosaccharides and carbohydrates. Depending on the particular application and type of enzyme analysed, different enzyme substrates options provide a more suitable solution. Megazyme can now provide an analytical solution for almost any carbohydrate hydrolase enzyme assay application.
1. Dyed Polysaccharides Page 3 a) Soluble substrates b) Insoluble substrates c) Tablet substrates
Suitable for the specific measurement of endo-acting carbohydrate hydrolases Suitable for the analysis of crude enzymatic extracts or industrial preparations Enzymatic activity determined by conversion from absorbance using a standard curve provided Generally not suitable for automated analytical assays (filtration step required)
Insoluble substrates: Suitable for gel/agar plate screening. Suitable only for semi-quantitative assays. Tablet substrates: Insoluble substrate formulated in tablets for quantitative assays and end-user convenience
2. Colourimetric Oligosaccharides Page 6 a) Blocked oligosaccharides b) Non-blocked oligosaccharides c) Colourimetric monosaccharides
Suitable for the measurement of either endo-acting or exo-acting carbohydrate hydrolases Chemically defined substrates: no variability between substrate batches 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)
Blocked oligosaccharides: For the analysis of crude enzymatic extracts or industrial preparations Non-blocked oligosaccharides: Suitable only for the analysis of purified enzymes
3. Carbohydrates Page 10 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 substrate solution
Polysaccharides: Native substrates of the enzymes being analysed Oligosaccharides: Chemically defined substrates, no variability between substrate batches
2 1. 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 polysaccharide or a partially depolymerised polysaccharide, to obtain substrates that are supplied as a fine powder. The endo-acting hydrolase 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 Suitable for the analysis of crude enzyme extracts The substrate is water soluble and is readily recognised and Suitable for the analysis of purified enzymes hydrolysed by an endo-acting hydrolase. The reaction is terminated by the addition of a precipitant solution (e.g. ethanol) wherein Chemically defined substrate high molecular weight, partially hydrolysed substrate fragments Suitable for automation (centrifugation required) precipitate from solution. The suspension is mixed thoroughly, Suitable for gel/agar plate activity screening centrifuged, and the colour in the solution is measured using a spectrophotometer. The enzymatic activity is determined using Quantitative assay a standard curve provided. Soluble chromogenic substrates are Suitable for endo-acting enzymes suitable for quantitative assays and can also be used in gel/agar Suitable for exo-acting enzymes plate activity screenings
Enzyme Activity Cat. No. Product α-Amylase S-RSTAR Red Starch endo-1,5-α-Arabinanase S-RDAR Red Debranched Arabinan (sugar beet) endo-Cellulase / endo-1,4-β-Glucanase S-ACMCL Azo-CM-Cellulose (liquid) S-ACMC Azo-CM-Cellulose (powder) S-AZXG Azo-Xyloglucan (tamarind) Lichenase / endo-b-Glucanase S-ABG100 Azo-Barley Glucan endo-Fructanase / endo-Inulinase S-AZFR5 Azo-Fructan S-AZFRXOI Azo-Fructan plus exo-Inulinase Pullulanase / Limit-dextrinase S-RPUL Red Pullulan endo-1,4-β-Mannanase S-ACGLM Azo-Carob Galactomannan endo-1,4-b-Galactanase S-AGALP Azo-Galactan (potato) endo-Protease S-AZCAS Azo-Casein (sulphanilamide dyed) endo-Rhamnogalacturonan hydrolase / lyase S-AZRH AZ-Rhamnogalacturonan endo-1,4-b-Xylanase S-AWAXL Azo-Wheat Arabinoxylan (liquid) S-AWAXP Azo-Wheat Arabinoxylan (powder) S-AXBL Azo-Xylan (birchwood) (liquid) S-AXBP Azo-Xylan (birchwood) (powder)
3 b) Insoluble dyed substrates Prepared by dyeing and crosslinking soluble polysaccharides. The Suitable for the analysis of crude enzyme extracts hydrated insoluble substrate forms gelatinous particles which are Suitable for the analysis of purified enzymes readily recognised and hydrolysed by an endo-acting hydrolase.The Chemically defined substrate partial hydrolysis of insoluble chromogenic substrates releases water Suitable for automation (filtration required) soluble dyed substrate fragments. The reaction is terminated by adding an alkaline solution to stop enzyme activity and the reaction Suitable for gel/agar plate activity screening slurry is filtered or centrifuged. The colour released in solution Quantitative assay is measured using a spectrophotometer and the colour intensity Suitable for endo-acting enzymes is directly related to enzyme activity. Insoluble chromogenic substrates are recommended for semi-quantitative assays. These substrates Suitable for exo-acting enzymes are provided in both granular and fine particle size [e.g. I-AZAMY (granular) and I-AZAMYF (finely milled)]. The finely milled substrates are much more useful for use 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.
Enzyme Activity Cat. No. Product a-Amylase I-AZAMY AZCL-Amylose NEW The use of agar plates containing I-AZAMYF AZCL-Amylose (fine) xanthan gum 0.5 % w/v avoids NEW I-RCLAMYF RedCL-Amylose (fine) settling of the insoluble dyed substrates (right) endo-1,5-α-Arabinanase I-AZDAR AZCL-Arabinan (debranched) endo-Cellulase I-AZCEL AZCL-HE-Cellulose I-AZCELF AZCL-HE-Cellulose (fine) NEW I-RCLCELF RedCL-HE-Cellulose (fine) I-ACELL Azo-α-Cellulose I-AAVIC Azo-Avicel I-AZXYG AZCL-Xyloglucan NEW I-AZXYGF AZCL-Xyloglucan (fine) NEW I-RCLXYGF RedCL-Xyloglucan (fine) NEW endo-Chitosanase I-AZCHAN AZCL-Chitosan NEW I-AZCHANF AZCL-Chitosan 1 (fine) endo-1,6-α-Dextrinase I-AZDEX AZCL-Dextran NEW I-AZDEXF AZCL-Dextran (fine) endo-1,4-b-Galactanase I-AZGLP AZCL-Galactan (potato) NEW I-AZGLPF AZCL-Galactan (potato) (fine) endo-1,3-b-Glucanase I-AZCUR AZCL-Curdlan NEW I-AZCURF AZCL-Curdlan (fine) I-RCLCURF RedCL-Curdlan (fine) I-AZPAC AZCL-Pachyman Lichenase / endo-b-Glucanase I-AZBGL AZCL-Barley b-Glucan NEW I-AZBGLF AZCL-Barley b-Glucan (fine) I-RCLBGLF RedCL-Barley b-Glucan (fine) 4 endo-1,4-b-Mannanase I-AZGMA AZCL-Galactomannan I-AZGMAF AZCL-Galactomannan (fine)NEW The use of insoluble dyed NEW substrates in agar plates containing I-RCLGMAF RedCL-Galactomannan (fine) xanthan gum 0.5% w/v allows endo-Protease I-AZCAS AZCL-Casein enzymatic activity detection more I-AZCOL AZCL-Collagen rapidly and at lower enzyme concentration (below) Pullulanase / Limit-Dextrinase I-AZPUL AZCL-Pullulan NEW I-AZPULF AZCL-Pullulan (fine) I-RCLPULF RedCL-Pullulan (fine) Rhamnogalacturonan hydrolase / lyase I-AZRHI AZCL-Rhamnogalacturonan 1 endo-1,4-b-D-Xylanase I-AZWAX AZCL-Arabinoxylan (Wheat) NEW I-AZWAXF AZCL-Arabinoxylan (Wheat) (fine) I-AZXBW AZCL-Xylan (Birchwood) I-RCLWAXF RedCL-Arabinoxylan (Wheat) (fine) c) Tablet substrates Megazyme supplies a range of enzyme tablet tests for ultimate Suitable for the analysis of crude enzyme extracts end-user convenience. The insoluble chromogenic substrates Suitable for the analysis of purified enzymes discussed above are formulated into tablets. From a procedural Chemically-defined substrate perspective, tablet tests operate in the same way as assays using Suitable for automation (filtration required) insoluble chromogenic substrates. The advantage of enzyme tablet tests is that the need to accurately weigh substrate quantities (and Suitable for gel/agar plate activity screening the error associated with this parameter) is removed. By using tablet Quantitative assay tests, it is possible to obtain quantitative determinations Suitable for endo-acting enzymes of endo-acting hydrolase activities. Suitable for exo-acting enzymes
Enzyme Activity Cat. No. Product α-Amylase T-AMZ Amylazyme T-AMZBG Amylazyme BG T-AMZRD Amylazyme Red T-AMZHY Amylazyme HY endo-1,5-α-Arabinanase T-ARZ Arabinazyme endo-Cellulase / endo-1,4-b-Glucanase T-CAF Cellazyme AF (40mg) T-CCZ Cellazyme C (60mg) T-CTZ Cellazyme T endo-Chitosanase T-CHZ Chitozyme endo-1,6-α-Dextranase T-DEXT α-Dextrazyme Lichenase / endo-b-Glucanase T-BGZ b-Glucazyme endo-1,4-b-Galactanase T-GLZ Galactazyme endo-1,3-b-Glucanase T-PAZ 1,3-b-Glucazyme T-CUR 1,3-b-Glucazyme HS Pullulanase / Limit-dextrinase T-LDZ Limit-Dextrizyme endo-1,4-b-Mannanase T-MNZ Mannazyme endo-Protease T-PRAK Protazyme AK T-PROL Protazyme OL Rhamnogalacturonan hydrolase / lyase T-RHAM Rhamnozyme endo-1,4-b-Xylanase T-PSYL Psyllazyme T-XAF Xylazyme AF (40mg) T-XAX Xylazyme AX (60mg) T-XYZ Xylazyme (100mg)
5 2. 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 colourimetric substrates Suitable for the analysis of crude enzyme extracts Blocked colourimetric substrates for the measurement of carbohydrate Suitable for the analysis of purified enzymes hydrolase activity are exclusively offered by Megazyme and are an excellent alternative to dyed polysaccharides for the quantitative Chemically-defined substrate measurement of endo-acting hydrolase activity in crude enzyme Suitable for automation (no filtration required) preparations. No variability between substrate batches occurs Suitable for gel/agar plate activity screening due to the chemically-defined molecular structures of these substances. The blocking group at the non-reducing end of the Quantitative assay substrate prevents the activity of exo-acting enzymes present Suitable for endo-acting enzymes in a crude mixture and their structure makes them absolutely specific targets for the endo-acting enzyme being analysed. These Suitable for exo-acting enzymes substrates are usually available as components within assay kits and are used in enzyme-coupled assay protocols. 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). The reaction is terminated by adding an alkaline solution to stop enzyme activity, the colour release in solution is measured using a spectrophotometer and the colour intensity is directly related to enzyme activity.
α-AMYLASE SUBSTRATES
K-CERA α-Amylase Assay Kit (Ceralpha method) K-AMYLSD α-Amylase SD Assay Kit (High sensitivity method) R-CAAR4 α-Amylase Reagent (Ceralpha) R-AMHR4 Amylase HR Reagent O-BPNPC7 Blocked 4-nitrophenyl-α-maltoheptaoside
CELLULASE / endo-1,4-b-GLUCANASE SUBSTRATES
K-CellG5 Cellulase Assay Kit (CellG5 method) K-CellG3 Cellulase Assay Kit (CellG3 method) R-CELLFLR Blocked 4-methylumbelliferyl-b-cellotrioside (Cellafluor reagent) O-B4MUG3 Blocked 4-methylumbelliferyl-b-cellotrioside O-BNAPG3 Blocked b-naphthyl-b-cellotrioside
6 LICHENASE / endo-b-GLUCANASE SUBSTRATES
K-MBG4 Malt b-glucanase / Lichenase Assay Kit (MBG4 method)NEW
PULLULANASE / LIMIT-DEXTRINASE SUBSTRATES
K-PullG6 Pullulanase / Limit-dextrinase Assay Kit (PullG6 method)
endo-1,4-b-XYLANASE SUBSTRATES
K-XylX6 endo-Xylanase Assay Kit (XylX6 method)NEW
b) Non-blocked colourimetric substrates Suitable for the analysis of crude enzyme extracts Non-blocked colourimetric substrates are generally used for the quantitative Suitable for the analysis of purified enzymes measurement of endo-acting hydrolase activity in purified enzyme Chemically-defined substrate preparations. The lack of a blocking-group makes these substrates unsuitable for the analysis of crude enzymatic mixtures because of Suitable for automation (no filtration required) the likely presence of competing exo-acting enzymes. In non-blocked Suitable for gel/agar plate activity screening oligosaccharide substrates, an oligosaccharide chain of defined length Quantitative assay is covalently linked to either a colourimetric (4-nitrophenol or 2-chloro-4- nitrophenol) or a fluorometric (4-methylumbelliferone) group. Purified Suitable for endo-acting enzymes endo-acting enzymes have very specific active site requirements. The Suitable for exo-acting enzymes 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.
7 CELLULASE / endo-1,4-b-GLUCANASE SUBSTRATES
2-Chloro-4-Nitrophenyl-b-Cello-Oligosaccharides
O-CPNPG2 DP2 2-Chloro-4-nitrophenyl-β-cellobioside O-CPNPG3 DP3 2-Chloro-4-nitrophenyl-β-cellotrioside O-CPNPG4 DP4 2-Chloro-4-nitrophenyl-β-cellotetraoside O-CPNPG5 DP5 2-Chloro-4-nitrophenyl-β-cellopentaoside
4-Methylumbelliferyl-b-Cello-Oligosaccharides
O-4MUG2 DP2 4-Methylumbelliferyl-β-cellobioside O-4MUG3 DP3 4-Methylumbelliferyl-β-cellotrioside O-4MUG4 DP4 4-Methylumbelliferyl-β-cellotetraoside O-4MUG5 DP5 4-Methylumbelliferyl-β-cellopentaoside
LICHENASE / endo-(1,3:1,4)-b-GLUCANASE SUBSTRATES
O-CNPBG3 DP3 2-Chloro-4-nitrophenyl-β-(1,3:1,4)-glucotrioside O-CNPBG4 DP4 2-Chloro-4-nitrophenyl-β-(1,3:1,4)-glucotetraoside O-4MUBG3 DP3 4-Methylumbelliferyl-β-(1,3:1,4)-glucotrioside
endo-1,3-β-GLUCANASE SUBSTRATES
4-Nitrophenyl-b-Laminari-Oligosaccharides
O-PNPLAM2 DP2 4-Nitrophenyl-β-laminaribioside O-PNPLAM3 DP3 4-Nitrophenyl-β-laminaritrioside O-PNPLAM4 DP4 4-Nitrophenyl-β-laminaritetraoside
4-Methylumbelliferyl-b-Laminari-Oligosaccharides
O-4MULAM2 DP2 4-Methylumbelliferyl-β-laminaribioside O-4MULAM3 DP3 4-Methylumbelliferyl-β-laminaritrioside O-4MULAM4 DP4 4-Methylumbelliferyl-β-laminaritetraoside
endo-1,4-b-MANNANASE SUBSTRATES
2-Chloro-4-Nitrophenyl-a-Manno-Oligosaccharides
O-CPNPAM2 DP2 2-Chloro-4-nitrophenyl-a-mannobioside O-CPNPAM3 DP3 2-Chloro-4-nitrophenyl-a-mannotrioside O-CPNPAM4 DP4 2-Chloro-4-nitrophenyl-a-mannotetraoside 4-Methylumbelliferyl-a-Manno-Oligosaccharides
O-4MUAM2 DP2 4-Methylumbelliferyl-a-mannobioside O-4MUAM3 DP3 4-Methylumbelliferyl-a-mannotrioside O-4MUAM4 DP4 4-Methylumbelliferyl-a-mannotetraoside
endo-1,4-β-XYLANASE SUBSTRATES
4-Nitrophenyl-β-Xylo-Oligosaccharides
O-PNPX2 DP2 4-Nitrophenyl-β-xylobioside O-PNPX3 DP3 4-Nitrophenyl-β-xylotrioside O-ONPX2 DP2 2-Nitrophenyl-β-xylobioside
4-Methylumbelliferyl-β-Xylo-Oligosaccharides O-4MUX2 DP2 4-Methylumbelliferyl-β-xylobioside O-4MUX3 DP3 4-Methylumbelliferyl-β-xylotrioside
8 c) Colourimetric substrates for 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. Once a soluble colourimetric monosaccharide substrate is hydrolysed by an exo-acting hydrolase, the colourimetric group is released in solution. The reaction is terminated by adding an alkaline solution to stop enzyme activity, the colour release in solution is measured using a UV spectrophotometer and the colour intensity is directly related to enzyme activity.
Suitable for the analysis of crude enzyme extracts Suitable for the analysis of purified enzymes Chemically-defined substrate Suitable for automation (no filtration required) Suitable for gel/agar plate activity screening Quantitative assay Suitable for endo-acting enzymes Suitable for exo-acting enzymes
Enzyme Activity Cat. No. Product
b-Amylase substrates K-BETA3 4-Nitrophenyl-β-maltotrioside + β-glucosidase (β-Amylase Kit)
R-BAMR3 4-Nitrophenyl-β-maltotrioside + β-glucosidase (β-Amylase Reagent)
O-PNPC3 4-Nitrophenyl-β-maltotrioside (β-Amylase Substrate)
O-NAPC3 b-Naphthyl-β-maltotrioside
Amyloglucosidase substrate R-AMGR3 4-Nitrophenyl-β-maltoside + β-glucosidase (Amyloglucosidase Assay Reagent)
β-N-Acetyl-D-Glucosaminidase O-4MUNAG 4-Methylumbelliferyl-β-N-acetyl-D-glucosaminide
O-PNPNAG 4-Nitrophenyl-β-N-acetyl-D-glucosaminide
α-L-Arabinofuranosidase O-PNPAF 4-Nitrophenyl-α-L-arabinofuranoside
α-Glucosidase O-PNPAG 4-Nitrophenyl-α-D-glucopyranoside
b-Glucosidase O-PNPBG 4-Nitrophenyl-b-D-glucopyranoside
α-Galactosidase O-PNPAGAL 4-Nitrophenyl-α-D-galactopyranoside
β-Galactosidase O-PNPBGAL O-D-galactopyranoside
α-glucuronidase O-PNPAGA 4-Nitrophenyl-α-D-glucuronide
α-galacturonidase O-PNPAGALA 4-Nitrophenyl-α-Nitrophenyl-α-D-galacturonide
β-galacturonidase O-PNPAGALA 4-Nitrophenyl-b-Nitrophenyl-α-D-galacturonide
β-Glucuronidase O-PNPBGA 4-Nitrophenyl-β-D-glucuronide
a-Fucosidase O-PNPAFC 4-Nitrophenyl-a-L-fucopyranoside
a-Xylosidase O-PNPAX 4-Nitrophenyl-a-D-xylopyranoside
β-Xylosidase O-PNPX 4-Nitrophenyl-β-D-xylopyranoside
a-Mannosidase O-PNPAM 4-Nitrophenyl-a-D-mannopyranoside
β-Mannosidase O-PNPBM 4-Nitrophenyl-β-D-mannopyranoside
Cellobiohydrolase O-PNPL 4-Nitrophenyl-β-lactoside
Chitobiosidase O-PNPCH2 4-Nitrophenyl-N-N1-acetyl-β-chitobioside
O-4MUCH2 4-Methylumbelliteryl-N-N1-diacetyl-β-chitobioside 9 3. Carbohydrates While dyed polysaccharide and colourimetric oligosaccharide-based assays are certainly the most convenient methods for the assay of hydrolytic enzymes, polysaccharides and oligosaccharides, as the native substrates encountered by carbohydrate hydrolases in nature, can give the truest insight into their mechanism of action, active site requirements, binding affinities and activities. a) Polysaccharides Traditionally, endo-acting carbohydrate hydrolases have been Suitable for the analysis of crude enzyme extracts measured using the native polysaccharide as the enzymatic substrate, followed by quantification of the increase in reducing sugar or decrease Suitable for the analysis of purified enzymes in viscosity of the substrate solution on hydrolysis. Reducing-sugar Chemically-defined substrate methods can be used for the assay of pure enzyme solutions but are Suitable for automation (no filtration required) unsuitable for use with crude enzyme extracts that may already contain reducing sugars. Viscosity reduction methods are specific Suitable for gel/agar plate activity screening for endo-hydrolase activity but are tedious to perform and require Quantitative assay specialist equipment. The fact that polysaccharides do not have Suitable for endo-acting enzymes a chemically defined structure could potentially lead to assay Suitable for exo-acting enzymes reproducibility issues between batches.
Enzyme Activity Cat. No. Product a-Amylase P-AMYL Amylose (potato)NEW P-BLDX b-Limit Dextrin endo-1,5-α-Arabinanase P-ARAB Arabinan (sugar beet) P-DBAR Debranched Arabinan (sugar beet) P-LARB Linear 1,5-α-L-Arabinan (sugar beet) P-CMLA CM-Linear 1,5-α-L-Arabinan (sugar beet) endo-Cellulase / endo-1,4-b-Glucanase P-CMC4M Carboxymethyl Cellulose 4M P-XYGLN Xyloglucan (tamarind) P-GLCML Glucomannan (konjac; low viscosity) P-GLCMH Glucomannan (konjac; high viscosity) endo-b-Chitinase P-CHITIN Chitin (colloidal) endo-1,6-α-Dextrinase P-DEXT DextranNEW endo-Fructanase P-FOS28 Fructooligosaccharides (DP2-8) P-INUL Inulin (DP2-60) endo-1,3-b-Galactanase P-ARGAL Arabinogalactan (larch wood) endo-1,4-b-Galactanase P-GALLU Galactan (lupin) P-GALPOT Galactan (potato) P-PGALU Pectic Galactan (lupin) P-PGAPT Pectic Galactan (potato) Lichenase / endo-b-Glucanase P-LICHN Lichenan (icelandic moss) P-MWBGS b-Glucan MW standard P-BGBL b-Glucan (barley; low viscosity) P-BGBM b-Glucan (barley; medium viscosity) P-BGBH b-Glucan (barley; high viscosity) P-BGOM b-Glucan (oat; medium viscosity) P-BGOH b-Glucan (oat; high viscosity) P-BGCFA b-Glucan CFA standard endo-1,3-b-Glucanase P-CURDL Curdlan
10 P-CMCUR CM-Curdlan P-PACHY Pachyman P-CMPAC CM-Pachyman P-BGYST b-Glucan (yeast; alkali soluble)
endo-1,4-b-Mannanase P-MANIV Mannan (ivory nut) P-MANCB Mannan (1,4-β-D-mannan) P-GALML Galactomannan (carob; low viscosity) P-GALMH Galactomannan (carob; high viscosity) P-GGMMV Galactomannan (guar; medium viscosity) P-GGMHV Galactomannan (guar; high viscosity) P-GGM21 Galactomannan (guar; galactose depleted; 21% galactose) P-GGM28 Galactomannan (guar; galactose depleted; 28% galactose) Pullulanase / Limit-Dextrinase P-PULLN Pullulan
P-PULLBH Pullulan (NaBH4 reduced) endo-Polygalacturonanase / Polygalacturonate lyase P-PGACT Polygalacturonic Acid (PGA) Rhamnogalacturonan hydrolase / lyase P-RHAM1 Rhamnogalacturonan I (potato) P-RHAGN Rhamnogalacturonan (soy bean) endo-1,4-β-Xylanase P-XYLNBE Xylan (beechwood; purified) P-RAXY Arabinoxylan (rye Flour) 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% arabinose) P-ADWAX26 Arabinoxylan (acid debranched; 26% arabinose) P-EDWAX30 Arabinoxylan (enzyme debranched; 30% arabinose) b) Oligosaccharides Defined oligosaccharide substrates are particularly useful for researchers Suitable for the analysis of crude enzyme extracts in the field of glycoscience. These molecules can be used to help characterise either endo- or exo-acting carbohydrate hydrolase enzymes, the activity Suitable for the analysis of purified enzymes of which can be measured through reducing sugar methods or HPLC/HPAEC- Chemically-defined substrate PAD analysis. A number of borohydride reduced oligosaccharides Suitable for automation (no filtration required) are also available. These are particularly useful substrates in reducing Suitable for gel/agar plate activity screening sugar assays where the native oligosaccharides, themselves being reducing sugars, can produce a high ‘blank’ absorbance value, thereby Quantitative assay reducing the sensitivity of the assay. Oligosaccharides can also be Suitable for endo-acting enzymes employed as building blocks for the chemical synthesis of either Suitable for exo-acting enzymes colourimetric substrates or inhibitors of enzymes of interest.
11 N-Acetyl-Chito-Oligosaccharides Substrates for characeterisation of endo-1,4-chitinase, chitobiosidase and N-acetyl-β-D-glucosaminidase
O-CHI2 DP2 Diacetyl-chitobiose O-CHI3 DP3 Triacetyl-chitotriose O-CHI4 DP4 Tetraacetyl-chitotetraose O-CHI5 DP5 Pentaacetyl-chitopentaose O-MTMT 63-α-D-Maltotriosyl-maltotriose 3 O-CHI6 DP6 Hexaacetyl-chitohexaose O-MTMTRD 6 -α-D-Maltotriosyl-maltotriitol (BH4 reduced)
Aldouronic Acids (from xylan) 1,5-α-L-Arabino-Oligosaccharides Substrates for characterisation of α-glucuronidase and endo-1,4-β- Substrates for the characterisation of endo-1,5-α-arabinanase and xylanase α-arabinofuranosidase
O-AMX Aldouronic acids mixture O-AMXR Aldouronic acids mixture (NaBH4 reduced) O-UXXR Aldotriouronic acid (borohydride reduced)NEW O-XUXXR Aldotetraouronic acid (borohydride reduced)NEW O-ABI DP2 Arabinobiose (syrup) Amylosaccharides (mixed-linkage) O-ATR DP3 Arabinotriose (syrup) Substrates for characterisation of limit-dextrinase, pullulanase, O-ATE DP4 Arabinotetraose (syrup) isopullulanase, neopullulanase, amyloglucosidase and α-glucosidase O-APE DP5 Arabinopentaose (syrup) O-AHE DP6 Arabinohexaose (powder) O-AHP DP7 Arabinoheptaose (powder) O-AOC DP8 Arabino-octaose (powder)
Branched Arabino-Oligosaccharides O-A5BMIX 22 32-di-α-L-Arabinofuranosyl-(1,5)-α-L-arabinotriose O-PAN Panose plus 32-α-L-arabinofuranosyl-(1,5)-α-L-arabinotetraose
O-A4B 32-α-L-Arabinofuranosyl-(1,5)-α-L-arabinotriose
Cello-Oligosaccharides Substrates for the characterisation of endo-1,4-β-glucanase, cellobiohydrolase, β-glucosidase and lipid polysaccharide O-IPAN Isopanose monooxigenase (LPMO)
O-CTR DP3 Cellotriose O-CTE DP4 Cellotetraose O-CPE DP5 Cellopentaose O-CHE DP6 Cellohexaose
O-GMT 63-α-D-Glucosyl-maltotriose Borohydride Reduced Cello-Oligosaccharides Substrates for the characterisation of endo-1,4-β-glucanase, cellobiohydrolase and β-glucosidase
O-CTRRD DP3 1,4-b-D-Cellotriitol O-CTERD DP4 1,4-b-D-Cellotetraitol O-CPERD DP5 1,4-b-D-Cellopentaitol
3 O-CHERD DP6 1,4-b-D-Cellohexaitol O-GMH 6 -α-D-Glucosyl-maltotriosyl-maltriose 12 1,3:1,4-β-Gluco-Oligosaccharides Substrates for the characeterisation of lichenase, endo-(1,3:1,4)-β- glucanase and β-glucosidase
O-GGM5 63, 64-α-D-Galactosyl-mannopentaose
Galacto-Oligosaccharides Substrates for the characterisation of O-BGTRIA 1,3:1,4-β-Glucotriose A β-galactosidase
O-BGTRIB 1,3:1,4-β-Glucotriose B O-GBI 1,3(4)-b-D-Galactobiose mix
Galactosyl-Sucrose Oligosaccharides Substrates for the characterisation of α-galactosidase
O-BGTETB 1,3:1,4-β-Glucotetraose B
O-BGTETC 1,3:1,4-β-Glucotetraose C O-VER Verbascose
Fructo-Oligosaccharides Gluco-Manno-Oligosaccharides Substrates for the characeterisation of endo-inulinase, exo-inulinase Substrates and standards for glucomannan degrading enzymes and fructanase
O-GMMBI Glucosyl-D-mannose plus, mannobiose
O-KTR DP3 1-Kestose O-KTE DP4 1,1-Kestotetraose O-GMMTR 1,4-β-D-Glucosyl-D-mannobiose plus 1,4-β-D- O-KPE DP5 1,1,1-Kestopentaose Cellobiosyl-D-mannose O-INU3 DP3 InulotrioseNEW 1,3-β-D-Gluco-Oligosaccharides Substrates for the characterisation of endo-1,3-β-glucanase Galacto-Manno-Oligosaccharides (laminarinase), exo-1,3-β-glucanase and β-glucosidase Substrates for the characterisation of endo-1,4-β-mannanase and α-galactosidase
O-LAM2 DP2 Laminaribiose O-LAM3 DP3 Laminaritriose O-LAM4 DP4 Laminaritetraose O-GM3 61-α-D-Galactosyl-mannotriose O-LAM5 DP5 Laminaripentaose O-LAM6 DP6 Laminarihexaose
Gluco-disaccharides (Various Linkages) Substrates for the characterisation of b-glucosidase and a-glucosidase O-GENT GlcβI,6GIc GentiobioseNEW O-LAM2 Glcb1,3Glc Laminaribiose O-SOPH Glcb1,2Glc SophoroseNEW O-GMM3 61-α-D-Galactosyl-mannobiose plus mannotriose O-IMO2 Glca1,6Glc IsomaltoseNEW
13 Isomalto-Oligosaccharides 1,4-β-D-Xylo-Oligosaccharides Substrates for the characterisation of endo-1,6-a-dextranase Substrates for the characterisation of endo-1,4-β-xylanase and and oligo-a-1,6-glucosidase β-xylosidase
O-XBI DP2 Xylobiose O-IMO2 DP2 IsomaltoseNEW O-XTR DP3 Xylotriose O-IMO3 DP3 IsomaltotrioseNEW O-XTE DP4 Xylotetraose O-IMO4 DP4 IsomaltotetraoseNEW O-XPE DP5 Xylopentaose O-IMO5 DP5 IsomaltopentaoseNEW O-XHE DP6 Xylohexaose O-XBIRD DP2 1,4-β-D-Xylobiitol Malto-oligosaccharides O-XTRRD DP3 1,4-β-D-Xylobiitol Substrates for the characterisation of a-amylase and b-amylase Arabino-Xylo-Oligosaccharides Substrates for the characterisation of α-arabinofuranosidase and endo-1,4-β-xylanase
O-MAL3 DP3 MaltotrioseNEW O-A3X 32-α-L-Arabinofuranosyl-xylobiose (A3X) O-MAL4 DP4 MaltotetraoseNEW
1,4-β-D-Manno-Oligosaccharides Substrates for the characterisation of endo-1,4-β-mannanase and β-mannosidase
O-A2XX 23-α-L-Arabinofuranosyl-xylotriose (A2XX)
O-MBI DP2 Mannobiose O-MTR DP3 Mannotriose O-MTE DP4 Mannotetraose O-MPE DP5 Mannopentaose O-MHE DP6 Mannohexaose
Xyloglucan Derived Oligosaccharides O-XA3XX 33-α-L-Arabinofuranosyl-xylotetraose (XA3XX) Substrates for the characterisation of α-xylosidase and endo-1,4-β- glucanase
O-IPRM Isoprimeverose (xyloglucan derived)
O-XAXXMIX 33-α-L- plus 23-α-L-Arabinofuranosyl-xylotetraose (XA3XX/XA2XX) O-XCBIR Xylosyl-cellobiose (borohydride reduced)
O-A23XX 23-,33-di-α-L-Arabinofuranosyl-xylotriose O-X3G4 Xyloglucan heptasaccharide (X Glc ) 3 4 (A2+3XX) O-X3G4R Heptasaccharide (X3Glc4; borohydride reduced) O-XGHON Xyloglucan (Hepta + octa + non asaccharides) O-XGHDP Higher DP xyloglucan oligosaccharides 14 www.megazyme.com
Bray Business Park, Bray, Co. Wicklow, A98 YV29, Ireland.
T + 353 1 286 1220
55 E. Monroe Street, Suite 3800, Chicago, Illinois, 60603, USA.
T + 1 312 212 4361
ISO 9001:2008 Registered April 2017 www.megazyme.com