NATARAJ at VM.SC.EDU wrote:
: 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.
Looks like you cloned the wrong gene.