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Enzyme Substrates

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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 endo-Acting Carbohydrate Hydrolase Substrates colourimetric Dyed Polysaccharide Substrates Carbohydrate Substrates Oligosaccharide Substrates Enzyme Activity Soluble Insoluble Tablet Blocked Non-blocked Polysaccharide Oligosaccharide α-Amylase ✓ ✓ ✓ ✓ ✓ ✓ endo-1,5-α-Arabinanase ✓ ✓ ✓ ✓ ✓ Cellulase / endo-1,4-β-Glucanase 1 ✓ ✓ ✓ ✓ ✓ ✓ ✓ endo-Chitosanase / endo-1,4-β-Chitinase ✓ ✓ ✓ ✓ ✓ endo-1,6-α-Dextranase ✓ ✓ ✓ ✓ endo-Fructanase / endo-Inulinase ✓ ✓ ✓ endo-1,4-β-Galactanase ✓ ✓ ✓ ✓ endo-1,3-β-Glucanase ✓ ✓ ✓ ✓ ✓ Lichenase /endo-1,3:1,4-β-Glucanase ✓ ✓ ✓ ✓ ✓ ✓ ✓ endo-1,4-β-Mannanase ✓ ✓ ✓ ✓ ✓ ✓ Pullulanase / Limit-dextrinase ✓ ✓ ✓ ✓ ✓ ✓ Rhamnogalacturonan hydrolase / lyase ✓ ✓ ✓ ✓ endo-1,4-β-Xylanase ✓ ✓ ✓ ✓ ✓ ✓ ✓ endo-Protease ✓ ✓ ✓ 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-β-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-β-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-β-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 α-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-β-Galactanase I-AZGLP AZCL-Galactan (potato) NEW I-AZGLPF AZCL-Galactan (potato) (fine) endo-1,3-β-Glucanase I-AZCUR AZCL-Curdlan NEW I-AZCURF AZCL-Curdlan (fine) I-RCLCURF RedCL-Curdlan (fine) I-AZPAC AZCL-Pachyman Lichenase / endo-β-Glucanase I-AZBGL AZCL-Barley β-Glucan NEW I-AZBGLF AZCL-Barley β-Glucan (fine) I-RCLBGLF RedCL-Barley β-Glucan (fine) 4 endo-1,4-β-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-β-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
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