I am working with production of cellulolytic enzymes from fungus ( such as Aspergillus sp) and I want to perform Endoglucanase, Exoglucanase and β-Glucosidase Zymograms. I didn't have problems performing the endoglucanase, I used CMC 0.05% and congo red as revelator, I can see 5 different bands from 25 to 40 KDa. But with the Exoglucanase and β-Glucosidase I revised the methods: for exoglucanase, I can use fluorescent substrates such as 4-Methylumbelliferyl β-D-cellobioside in a Native PAGE to see the specific activity. For β-glucosidase I can use another fluorescent substrate such as 4-Methylumbelliferyl β-D-glucopyranoside or Esculin (Kwon et.al, 1994) to see the band.
The problem is that the fluorescent substrates are expensive, and the delivery times are more than two months.
Any other suggestions about how to perform the zymogram?
I have done with different dyes, like remazole brilliant blue, phenol red, and tripan blue, like suggest,in the paper "Comparison of Dyes for Easy Detection of Extracellular Cellulases in Fung" by Hwan Joon et.al. whithout favorable results.
I appreciate the problems caused by expense and by delay in delivery but the very high fluorescence of methylumbelliferone gives great sensitivity for the location of the enzyme bands that is unlkely to be matched by any other substrate. You may have to grit your teeth and bare the cost.
Peter Butterworth
Ok... thanks for all suggest... I believe I need to save money to buy the fluorescent substrates or Esculin.
The best way to differentiate exo- and endo-glucanase is TLC. Exo-acting glucanases can deal with Avicel (crystalline cellulose) as substrate but endo-acting enzymes cannot. See the attachment for methods.
Thanks for all responses and papers, all are being really useful.
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