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

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Abstract

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 (pNA)], the enzyme preferred d-Leu-pNA and d-Phe-pNA 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-pNA 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-NH2 from d-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from d-Leu-pNA 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.

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Acknowledgments

This study was partly supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Author notes

  1. Junichiro Marui

    Present address: Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686, Japan

Authors and Affiliations

  1. National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan

    Mayumi Matsushita-Morita, Hiroyuki Nakagawa, Sawaki Tada, Junichiro Marui, Ryota Hattori, Satoshi Suzuki & Ken-Ichi Kusumoto

  2. Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Youhei Yamagata & Michio Takeuchi

  3. Gifu R&D Center, Amano Enzyme, Inc., 1-6 Technoplaza, Kakamigahara, Gifu, 509-0109, Japan

    Hitoshi Amano

  4. Research Institute, Gekkeikan Sake Company Ltd., 101 Simotobakoyanagi-cho, Fushimi-ku, Kyoto, 612-8385, Japan

    Hiroki Ishida

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  1. Mayumi Matsushita-Morita
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Correspondence to Ken-Ichi Kusumoto.

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Matsushita-Morita, M., Nakagawa, H., Tada, S. et al. Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae . Appl Biochem Biotechnol 171, 145–164 (2013). https://doi.org/10.1007/s12010-013-0330-z

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