Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae

• Published in: Applied biochemistry and biotechnology Vol. 171; no. 1; pp. 145 - 164
• Main Authors: Matsushita-Morita, Mayumi, Nakagawa, Hiroyuki, Tada, Sawaki, Marui, Junichiro, Hattori, Ryota, Suzuki, Satoshi, Yamagata, Youhei, Amano, Hitoshi, Ishida, Hiroki, Takeuchi, Michio, Kusumoto, Ken-Ichi
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.09.2013; Springer; Springer Nature B.V
• Subjects: Amino acids; Aspergillus oryzae; Biochemistry; Biological and medical sciences; Biosynthesis; Biotechnology; Chemistry; Chemistry and Materials Science; Fundamental and applied biological sciences. Psychology; Peptides; Sodium chloride; Stereoselective aminopeptidase; Halophilicity; Amino acid; Leucine; Aminolysis; D-Leucine; Enzyme; Aminopeptidases; D-Amino acid; Fungi; Characterization; Stereoselectivity; Peptidases; Aspergillus oryzae; Aminoacid; D-Stereoselective aminopeptidase; Hydrolases; Fungi Imperfecti
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-013-0330-z

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae . The gene was overexpressed in A. oryzae , and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide ( p NA)], the enzyme preferred d -Leu- p NA and d -Phe- p NA as substrates. Therefore, we designated this gene as d - stereoselective aminopeptidase A ( damA ). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward d -Leu- p NA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0–11.0. DamA also exhibited aminolytic activity, producing d -Leu- d -Leu-NH 2 from d -Leu-NH 2 as a substrate. In the presence of 3.0 M NaCl, the amount of p NA liberated from d -Leu- p NA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a d -amino acid at the N-terminus as well as physiologically active peptides.