Hi everyone,
I try to purify an enzyme (bezoate dihydrodiol dehydrogenase) from
Rhodococcus opacus (Gram +).
I noticed that the optimal pH for this enzyme is 9, so I started an
anion exchange chromatography with buffer pH 8 because it was supposed
that 9 is too high. The chomatogram was nice and i got activity in a
fraction. The problem came afterwards when I tryed a Hydrofobic
Interaccion Chrom. I precipitated the protein pool with (NH4)2SO4 and
resuspended them with puffer pH8+salt. The activity was high so I did a
HIC (phenyl sepharose) with decreasing concentration of TrisHCl 25mM pH8
+ (NH4)2SO4 1M. I got a good chromatogram but no activity in any
fraccion. Then I tryed a butyl Q-sepharose column and also didnĄt work.
Last time I used a buffer pH 7,5 and I had again no activity.
Do you have any idea where the problem can be?
Thanks
Angela
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