【Megazyme】Enzyme Substrates:Dyed Polysaccharides

Dyed polysaccharides are useful substrates for the specific measurementof endo-acting hydrolases activity in crude plant extracts or industrialenzyme preparations.

In dyed polysaccharides, dye molecules are covalently attached to apolysaccharide or a partially depolymerised polysaccharide, to obtainsubstrates that are supplied either as a powder or in liquid form.

The endo-acting hydrolase enzymes that recognise and act on thesesubstrates release dyed fragments to an extent which is proportionalto their enzymatic activity and which can be calculated by convertingthe absorbance obtained using a standard curve provided.

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

Key features of Megazyme's soluble dyed substrates

√Suitable for analysis of crude enzyme extracts

√Suitable for analysis of purified enzymes

√Quantitative assay

√Suitable for gel/agar plate activity screening

√Suitable for endo-acting enzymes

×Chemically defined substrate

×Suitable for automation

×Suitable for exo-acting enzymes

Assay procedure for soluble dyed substrates

1.The substrate is water soluble and is readily recognised and hydrolyseo 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

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

√Suitable for analysis of purified enzymes

√Suitable for gel/agar plate activity screening

√Suitable for endo-acting enzymes

×Quantitative assay

×Chemically defined substrate

×Suitable for automation

×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 partia hydrolysis ofinsoluble 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.

Our range of insoluble dyed 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 guantitative determinations of endo-acting hvdrolase 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

√Suitable for analysis of purified enzymes

√Quantitative assay

√Suitable for endo-acting enzymes

×Chemically defined substrate

×Suitable for automation

×Suitable for gelagar plate activity screening

×Suitable for exo-acting enzymes