I have zone of clearance on Collidal chintin plate but I am not getting enzyme activity when inoculated from broth. I am using DMAB method. Is there different assay methods to differentiate between endochitinase and exochitinase. should I use different method or any other analytical method. Please suggest?
Does your "zone of clearance" have a ring around the outermost perimeter (kind of like a wall)? You may not be cleaving the chitin but agglutinating it instead.
Try diluting your starting material and assaying for a longer period - lectins/agglutinins will dilute out, whereas enzymatic activity will still be detectable (given enough time)...
Is enzyme activity regulated so that expression only occurs in the presence of chitin or similar substrates? I imagine you don't have the colloidal chitin in broth.
Dear Micheal and Lorenzo...Thanks for your suggestion
@Micheal: We are putting Chitin in the broth...minimal media with yeast extract....standard protocol
@Lorenzo...We didn't observe the outermost parameter....I will definately have a closer look..Thanks
However unfortunately we are not getting the production also....we are not able to see any sugar production in supernatant also...we are using DMAB method for estimation
Vikas,
Another thought has occurred to me: in the colloidal chitin plate you add several metal ions that are co-factors for 'the usual' chitinases. Are these metal ions also present in your broth?
DMAB should detect amidated sugars just fine. I'm assuming that the DMAB assay has worked properly in your lab before...
Lorenzo
Lorenzo,
Ya its the same media.....this is the first time we are using this method...and the DMAB method is working with the standard xylose curve...
Is it possible that its an endochitinase and we do see the sugars monosaccharides and there fore DMAB is not working
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