The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx18E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using l-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.
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This research was supported in part by grants from the Noda Institute for Scientific Research. We thank Mr. Y. Nishimoto and Mr. R. Nakahigashi of Nagase for the helpful advice.
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Hatanaka, T., Arima, J., Uraji, M. et al. Characterization, cloning, sequencing, and expression of an aminopeptidase N from Streptomyces sp. TH-4. Appl Microbiol Biotechnol 74, 347–356 (2007). https://doi.org/10.1007/s00253-006-0669-y