I am running a purification of cellulase from malted sorghum. After ammonium sulphate precipitation, a pH and buffer stability test showed the enzyme was more stable in acetate or citrate buffers at pH 5 or 5.5 but looses activity in phosphate buffer at pH 6.5 and above. But when I applied it on DEAEcellulose with acetate buffer at pH 5.0, it did not bind but other proteins of non-interest bound to the column. Also it behaved the same way when I changed to CMsepharose with the same buffer at the same pH. I don't want to risk loosing the stability of the enzyme by manipulating the pH to alkaline range with a different buffer. Please why did it behave this way and what should I do? Thank you all.
DEAE does not do a lot at pH 5. Try pH 7 - 8. Try the stability of your enzyme in a different buffer than phosphate; e.g., Tris. Quite a lot of enzymes actively bind phosphate and it can do strange things to them.
That your enzyme is not stable at a pH of 6.5 to 7.5 is rather unusual; after all the cellular pH is pH 7 or even slightly above. You could try adding glycerol up to 4%.
How much of the ammonium sulfate is still with your enzyme? Make sure the conductivity of your chromatography load sample is not too high.
Check if you can find some information about your enzyme in the enzyme database BRENDA (https://www.brenda-enzymes.org).
Thank you Mr. Achim, I appreciate your response. The enzyme is not carrying any ammonium sulphate load. The ion exchange chromatography was done after gel filtration with sephadex g-100. Anyway, I ve only tried phosphate buffer at pH 6.5 to 6.8.
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