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Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

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An Erratum to this article was published on 27 November 2011

An Erratum to this article was published on 27 November 2011

Abstract

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.

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Acknowledgments

This study was partially supported by the Program for Promotion of Basic Research for Innovative Bioscience (PROBRAIN). We would like to thank Dr. Yoshio Tanaka at Amano Enzyme Inc. for the helpful discussions and support for the peptide hydrolysis analysis.

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Authors and Affiliations

  1. Applied Microbiology Division, National Food Research Institute, 2-1-12 Kan-nondai, Tsukuba, Ibaraki, 305-8642, Japan

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

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

    Hitoshi Amano

  3. Gekkeikan Sake Co Ltd., 247 Minamihama-cho, Fushimi-ku, Kyoto, Kyoto, 612-8660, Japan

    Hiroki Ishida

  4. Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Youhei Yamagata & Michio Takeuchi

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  1. Junichiro Marui
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  2. Mayumi Matsushita-Morita
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Correspondence to Ken-Ichi Kusumoto.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s00253-011-3690-8

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Marui, J., Matsushita-Morita, M., Tada, S. et al. Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji). Appl Microbiol Biotechnol 93, 655–669 (2012). https://doi.org/10.1007/s00253-011-3610-y

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