3 replies to this topic

[schroddycat]

Dear all, appreciate your comments here.

I'm currently extracting GAD enzyme (Glutamate decarboxylase) by doing enzymatic lysis onto the fungus Aspergillus Oryzae.The lytic enzymatic cocktail was Yatalase, which composing of Chitinase, Chitobiase, Chitosanase, beta-1,3-glucanase and Protease.

I used the cocktail (10 mg/ml) to lyse 5, 10, 20, 30, 40, 50, & 60% w/v of fungal concentration. After incubation time (60 mins), the supernatant was collected for determining total protein (Bradford reagent) and enzymatic activity (GABA Assay).

Here's my finding: total protein increased proportionately with fungal cell concentration. However, total activity and specific activity of GAD enzyme decreased from 30% w/v fungal cell concentration. IMO, I thought total activity of GAD would increase with total protein i.e. more proteins released from fungal cells, more GAD collected as well. Is this result usual or unusual? What phenonmenon caused it?

Tl;dr:

Fungus (5, 10, 20, 30, 40, 50, & 60% w/v) Undergo enzymatic lysis. Enzyme of interest: GAD

Total protein: increases
Total activity of GAD: decrease
Specific activity of GAD: decrease



Not sure how to interpret the result.

Edited by schroddycat, 02 September 2012 - 12:48 AM.

[bob1]

Proteasese?  If you have enough proteases it will swamp out the inhibitors you should have present.

[prabhubct]

You  can try cocktail (10 mg/ml) in increasing order 10,20,30,........... for 5, 10, 20, 30, 40, 50, & 60% w/v of fungal concentration.

[ascacioc]

Besides the proteases, it can also happen that too much enzyme self-inhibits the reaction. In the case of Taq polymerase it is so i.e. I had to dilute the enzyme several times after purification in order for me to get an activity. Check the literature for your enzyme self-inhibition. Or maybe there is a component in your enzyme preparation that inhibits your protein and by getting diluted while the enzyme gets diluted, it also allows an activity. Also check the literature for known inhibitors of your reaction.

Andreea