Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus

Toshinobu Arai, Atsushi Noguchi, Eriko Takano, Kuniki Kino

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min-1 mg-1·protein and 136 ± 6 nmol min-1 mg-1·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.

    Original languageEnglish
    Pages (from-to)382-387
    Number of pages6
    JournalJournal of Bioscience and Bioengineering
    Volume115
    Issue number4
    DOIs
    Publication statusPublished - 2013 Apr

    Fingerprint

    amidase
    Acidic Amino Acids
    Dipeptides
    Amino acids
    aspartyl-phenylalanine
    Proteins
    glutaminyl-glycine
    Aspartame
    Asparagine
    Glutamine
    alanylglutamine
    Amidohydrolases
    Amino Acids
    Sweetening Agents
    Acids
    Adenosinetriphosphate
    Valine
    Ligases
    Aspartic Acid
    Yeast

    Keywords

    • Aspartame
    • Functional peptide
    • L-Amino acid ligase
    • Peptide synthesis
    • Protein N-terminal amidase

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology
    • Bioengineering

    Cite this

    Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus. / Arai, Toshinobu; Noguchi, Atsushi; Takano, Eriko; Kino, Kuniki.

    In: Journal of Bioscience and Bioengineering, Vol. 115, No. 4, 04.2013, p. 382-387.

    Research output: Contribution to journalArticle

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