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Overexpression and Characterization of an Extracellular Leucine Aminopeptidase from Aspergillus oryzae

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Abstract

Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5–11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.

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Acknowledgment

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

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Authors and Affiliations

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

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

  2. Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, 3-8-1, Harumi-cho, Fuchu, Tokyo, 183-0057, 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

  5. Department of Fermentation Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan

    Yutaka Kashiwagi

Authors
  1. Mayumi Matsushita-Morita
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  2. Sawaki Tada
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  8. Hitoshi Amano
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  9. Hiroki Ishida
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  11. Yutaka Kashiwagi
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  12. Ken-Ichi Kusumoto
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Correspondence to Ken-Ichi Kusumoto.

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Matsushita-Morita, M., Tada, S., Suzuki, S. et al. Overexpression and Characterization of an Extracellular Leucine Aminopeptidase from Aspergillus oryzae . Curr Microbiol 62, 557–564 (2011). https://doi.org/10.1007/s00284-010-9744-9

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  • DOI: https://doi.org/10.1007/s00284-010-9744-9

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