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?

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