Mg and pH optimum

Michael Cooley szcooley at chip.ucdavis.edu
Thu Oct 17 06:40:28 EST 1996


NATARAJ at VM.SC.EDU wrote:
: Hi,
: 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.
: Natarajan

Looks like you cloned the wrong gene.



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