I'm trying to separate cellulases from a crude extract, obtained from a Solid State fermentation of a Filamentous fungi with lignocellulosic waste. In a Zymogram we confirm the Molecular weight of the enzymes, and I have 2 bands with Endoglucanase activity about 35 and 45 KDa, and 1 band with activity of b- Glucosidase about 70KDa. The problem is the protein and the activity of both enzymes that are in at the same time and the same collected fraction. I also tried changing the flux speed and the size of the column, (Bigger and smaller) with similar results, and reducing the amount of sample to 1ml to 0.5ml .
Conditions of the experiment:
Resine : Biogel P-100 (Biorad) MW- Fractionation 5-100 KDa
Column : 1x50cm (35 ml Packed)
Flux speed : 1 ml/min with Acetate Buffer pH 4.8 100mM
Sample : 1 ml of Ajusted to 0.1mg/ml of total protein ( extract from SSF without particles)
Collected fractions : 3ml each
Operation temperature : 20ºC
The problem is the low resolution and the amount of protein and enzymatic activities are in the same time
Any suggestions?