Cloning and Nucleotide Sequencing of Prolyl Endopeptidase Gene from Aeromonas punctata subsp. punctata.

Prolyl endopeptidase activity was found in Aeromonas punctata subsp. Punctata. The genomic DNA was partially digested with EcoRI and the recovered 8-16 kb DNA fragments were inserted into the EcoRI site of plasmid pUC19, and were transformated into Escherichia coli DH5alpha. The resulted clones were screened by using Benzyloxycarbonyl-Gly-Pro-beta-naphthylamide, the specific substrate of prolyl endopeptidase and a positive clone was obtained. The 12 kb insertion fragment of recombinant plasmid was digested with HincII and subcloned. The PEP gene was found in the 3.5 kb HincII/EcoRI fragment. Nucleotide sequence of the gene was completely sequenced by Auto Sequencer. The complete gene consisted of 2 073 bp corresponding to 690 amino acid residues with a calculated molecular weight of 76 467 Da. The amino acid sequence was 92.3% 53.2% 33.5% 33.2% and 20.5% homologous to those of Aeromonas hydrophila, Flavobacterium meningosepticum, porcine brain, human lymphocytes and Pyrococcus furiosus respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser(538) Asp(622) and His(657).