Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans

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Abstract
A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans , the gene encoding the enzyme was cloned and expressed in Escherichia coli , and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis( p -nitrophenyl)phosphate with a K m of 2.9 ± 0.1 mM and a k cat of 879 ± 73 min −1 . The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli . The IS 1071 transposon insertion sequence was found downstream of pdeA .