Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

Aminopeptidase P (APP, or X-Pro aminopeptidase; EC 3.4.11.9) is a peptidase that specifically removes the N-terminal amino acids from peptides in which the penultimate residue is proline (5). Since the time an enzyme with the specificity of APP was first purified from Escherichia coli (24), APPs have been characterized from diverse sources, including bacteria (16), nematodes (15), insects (14), plants (10), and tissues from several mammalian species (9, 11). While the physiological role of APP in bacteria is unclear, mammalian APP is involved in the protein turnover of collagen and the regulation of biologically active peptides, such as substance P and bradykinin (5, 23, 26). It has been shown that APPs from a number of lactococcal strains may contribute to the abolition of bitterness during the ripening of cheese by participating in peptide degradation following release into the cheese matrix (17). To date, however, there have been no reports on the properties of an APP from either an archaeon or a hyperthermophile. With the availability of a generally applicable combination of conventional genetic engineering and genomic research techniques, the genome sequences of some hyperthermophilic microorganisms are of considerable biotechnological interest because of their heat-stable enzymes, and many extremely thermostable enzymes are being developed for biotechnological purposes (22). Furthermore, recent advances in the application of molecular biological tools to hyperthermophilic archaea, such as gene knockout techniques and efficient transformation systems, could facilitate the study of hyperthermophilic archaeal gene function and contribute to an understanding of the physiology of hyperthermophilic archaea. To facilitate the search for valuable and extremely thermostable enzymes and to help answer questions concerning the physiology of hyperthermophilic archaea grown at extremely high temperatures, we recently isolated a hyperthermophilic archaeon, Thermococcus sp. strain NA1 (S. S. Bae et al., unpublished data), and its whole genome sequence was determined (J.-H. Lee et al., unpublished data). Analysis of the genome information of Thermococcus sp. strain NA1 revealed an APP gene that is similar to those for other APPs. In the present study, the gene corresponding to an APP was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined.