title: Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme.
abstract: The aminopeptidase P gene in Escherichia coli HB101 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPP01, 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 hydroxyapatite. The purified enzyme was homogeneous as judged by disc-gel and SDS-gel electrophoreses. the enzyme was inhibited strongly by EDTA and slightly by p-chloromercuribenzoate, 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 min at pH 8.0, 50% remaining activity was observed at 50 degrees C. The enzyme was activated 3-fold by the addition of 5 microM 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].
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University.
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: N
authors.initials: N
authors.lastname: Murayama
authors.forename: T
authors.initials: T
authors.lastname: Honda
authors.forename: H
authors.initials: H
authors.lastname: Tone
authors.forename: D
authors.initials: D
authors.lastname: Tsuru
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D004262:DNA Restriction Enzymes; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase