Proline iminopeptidase gene from Xanthomonas campestris pv. citri.

Abstract

The pip gene coding for the proline iminopeptidase (Pip) of Xanthomonas campestris pv. citri was cloned in an Escherichia coli leuB strain using a selective medium containing the dipeptide D-Ala-L-Leu as the sole source of L-leucine. Nucleotide sequencing of this gene revealed a 939 bp open reading frame encoding a 312 amino acid protein (35 126 Da). The deduced amino acid sequence showed 47% identity with the Pip from Neisseria gonorrhoeae. A lacZ-pip fusion gene was overexpressed in E. coli under the control of the Plac promoter. The Pip of X. campestris hydrolysed L-prolyl-p-nitroanilide with the highest efficiency, but was also able to hydrolyse L-alanyl-p-nitroanilide and D-alanyl-p-nitroanilide. The molecular mass of Pip was found to be 35 kDa by SDS-PAGE and 120 kDa by gel filtration, suggesting that the active enzyme is a multimer.