I have cloned a phthalate hydrolase (~37 kDa) into a pMAL-c2x vector and overexpressed in E.coli BL21(DE3).

Induction condition - 28 degrees for 3h with 0.5mM IPTG.

This is a Mycolicibacterium derived esterase which can hydrolyze di-(2-ethylhexyl)phthalate (DEHP) to its byproducts mono-(2-ethylhexyl)phthalate (MEHP) and phthalic acid which can be evaluated with enzyme assays followed by TLC.

I have observed the transformation of DEHP with the protein (with both crude and pure recombinant protein) , however while repeating the process again (multiple times with same conditions), the protein seems to be non-functional with the same assay condition.

Buffer system used - Tris-HCl, pH-8.0

Can anyone suggest??

Thank you in advance.