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a) Soluble Dyed Substrates

1. The substrate is water soluble and is readily recognised and hydrolysed by an endo-acting hydrolase.

2. The reaction is terminated by the addition of a precipitant solution (e.g. 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
Red Starch	S-RSTAR	α-Amylase
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-Cellulase / endo-1,4-β-Glucanase
Azo-Xyloglucan (tamarind)	S-AZXG
Azo-Barley Glucan	S-ABG100	Lichenase / endo-b-Glucanase
Azo-Fructan	S-AZFR5	endo-Fructanase / endo-Inulinase
Azo-Fructan plus exo-Inulinase	S-AZFRXOI
Red Pullulan	S-RPUL	Pullulanase / 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 / lyase
Azo-Wheat Arabinoxylan (liquid)	S-AWAXL
Azo-Wheat Arabinoxylan (powder)	S-AWAXP	endo-1,4-b-Xylanase
Azo-Xylan (birchwood) (liquid)	S-AXBL
Azo-Xylan (birchwood) (powder)	S-AXBP

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.

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
1.
The hydrated insoluble substrate forms gelatinous particles which are readily recognised and hydrolysed by an endo-acting hydrolase. The partial hydrolysis of insoluble chromogenic substrates releases water soluble dyed substrate 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.

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-Chitosanase
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

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
 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-Dextranase
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