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J. Biochem, 1988, Vol. 104, No. 1 93-97
© 1988 Japanese Biochemical Society

research-article

Cloning and Expression of Aminopeptidase P Gene from Escherichia coli HB101 and Characterization of Expressed Enzyme1

Tadashi Yoshimoto, Nobuhiro Murayama, Takashi Honda, Hiroshi Tone and Daisuke Tsuru

School of Pharmaceutical Sciences, Nagasaki University Nagasaki, Nagasaki 852

The aminopeptidase P gene in Escherichia coli HB1O1 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPPO1, into the E. coli DH1 resulted in an 8-fold increase of aminopeptidase P activity as compared with that of the host. The enzyme was purified by series of chromatographies on DEAE-Sephadex, QAE-Sephadex, and hydroxy apatite. The purified enzyme was homogeneous as judged by disc-gel and SDS-gel electro phoreses. The enzyme was inhibited strongly by EDTA and slightly by p-chloromercuri benzoate, but was not affected by diisopropyl phosphorofluoridate, E-64, or iodoacetic acid. The optimum pH of the enzyme was 8.5. The enzyme was stable at pH 8 to 9. After incubation for 30 mm at pH 8.0, 50% remaining activity was observed at 50° The enzyme was activated 3-fold by the addition of 5 µM Mn2+ The molecular weight of the enzyme was estimated to be 50,000 and 200,000 by SDS-PAGE and gel filtration, respectively. The amino terminal amino acid was identified to be serine by Edman degradation, indicating that the enzyme is composed of a homo-tetramer. The enzyme hydrolyzed X-Pro bonds (X = amino acid) of peptides. These characteristics suggest that cloned aminopeptidase P is identical to APP-II reported by Yoshimoto et al. (Agric. Biol. Chem. 52(8), in press (1988).

This work was supported in part by the Research Fund of the Nagasaki Super Technology Development Association.


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