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?
It is likely that both proteins are joined by noncovalent interactions. My suggestion is to apply sample to a different type of chromatography, ion exchange or affinity for instance. If you want to keep trying by gel filtration might be appropriate to change the buffer in which the sample is located, and the elution buffer, perhaps a higher ionic strength would break the interactions between the two enzymes.
Size exclusion might not be the best approach as Esau has stated. If you need to use size exclusion, then increasing salt concentration to 500mM, or decreasing salt but adding a small amount of glycerol, ie about 5-10% may help separate things. Or adding substrate with the chromatography, might help separate. You would just need to empirically determine things. If the interaction is ionic, then salt will help. If the interaction is hydrophobic, then glycerol will help. You can also try a HPLC sizing column for better resolution. I used this with two similar proteins, and it worked well.
- Badges
- Science method