Loss of glucose during enzyme assay - (Nov/28/2011 )
I am trying to measure endoglucanase activity using the DNS detection method. This particular enzyme has a temp optima of 80 C - thermophilic. My solutions initially have glucose so I measure glucose levels before and after activity with CMC (carboxymethyl cellulose).
In my control I have noticed that ~20% of my initial glucose is gone after the reaction goes for 72 hours. I am wondering whether my glucose could be degrading from heat at 70 C? Or fermented? The incubation lasts this long because the small amount of enzyme I have to work with needs to release a fair amount of glucose to get above the sensitivity limit.
Reporting concern: if I say apprx 20% of my glucose is lost during the control reaction, can I factor that back into my actual sample? Trying to account for glucose lost (some how) and gained (released from CMC) seems dicey when using a total sugar assay.
Side notes: I have 0.1% sodium azide in my CMC at pH 5. I have tried Azo-CMC but was not sensitive and seemed to have interference from my background solution.
Any advice helpful, Thanks.
Have you thought about a MUG based assay instead? There is probably a MUG carbohydrate derivative that is a substrate for your enzyme, and is easily detectable as a fluorescent product in a plate assay.
There are reports using MUC (4-methylumbelliferyl B-D-cellobioside) but I've heard that extracts from soil matrices (which is what i'm doing) makes using fluorescence difficult due to quenching..?? I may have to try it though as I am out of ideas. Thanks phage