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Mg and pH optimum

Tue Oct 15 14:31:14 EST 1996

I am working on an amino-acid degrading enzyme from a soil bacterium. The pH
optimum for the activity in the crude bacterial extract is 6.8 and does not
require Mg for its activity.  I cloned the gene coding for enzyme and
overexpressed it in E.coli.  I purified the enzyme from  E.coli and did an
enzyme assay.  The pH optimum for the recombinant protein seems to 5.5 instead
of 6.8!  I tried adding back different metals and found that  adding Mg to
200mM concentrationshifts the peak activity to 7.5. My question is what causes
this discrepency in the pH optimum and how can Mg substitute whatever there
is in the crude extract that makes the enzyme behave differently.Thanks in
advance for your suggestions.

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