Characterization of a unique proline iminopeptidase from white-rot basidiomycetes Phanerochaete chrysosporium

Abstract

A putative gene encoding proline iminopeptidase (PchPiPA) was cloned from Phanerochaete chrysosporium BKM-F-1767 by RT-PCR and expressed successfully in Escherichia coli. The cDNA is 942 bp in length and encodes 313 amino acids. The recombinant enzyme was only able to hydrolyze Pro- pNA among the tested synthetic substrates. There is no activity detected toward Leu- pNA, Phe- pNA and Tyr- pNA, as well as GGG- pNA, SGR- pNA, AAV- pNA, AAPL- pNA, AAVA- pNA. And the recombinant enzyme could cleave the peptides derived from enzyme-hydrolytic natural proteins to release free lysine, which was confirmed using synthetic oligopeptides with lysine at N termini as substrate. The optimal pH and temperature for this enzyme were 8.0 and 45 °C, respectively. The catalytic activity was inhibited slightly by Mg 2+, Al 3+, Ca 2+, Fe 3+, Fe 2+ and Ba 2+; strongly by Ni 2+, Mn 2+ and Co 2+, and almost inactivated by Zn 2+, Cu 2+ and Hg 2+. In addition, the enzyme was not sensitive to EDTA-Na 2, as well as redoxes of DTT, β-ME and H 2O 2. The protease inhibitors of benzamidine hydrochloride and phenylmethyl sulfonyfluoride caused a moderate inhibition. The V max, K m and k cat toward Pro- pNA were 347.86 μmol min −1 mg −1, 2.15 mM and 218.10 S −1, respectively. The deduced catalytic triad of Ser 107, Asp 264 and His 292 was confirmed by site-directed mutagenesis because the individual replacement of Ser 107 to Asp, Asp 264 to Ala or His 292 to Leu led complete inactivation. Transcriptional analysis by RT-PCR showed that PchPiPA could be expressed under ligninolytic and non-ligninolytic conditions. Conclusively, it was suggested that the proline iminopeptidase may be a member of the proteolytic system in this fungus. The availability of recombinant protein may be potentially used in certain proteolytic processing.