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 degrees 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, beta-ME and H(2)O(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 mumol 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.
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