Synthesis of DL-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996

Kuniki Kino, Masaru Sato, Mariko Yoneyama, Kotaro Kirimura

    Research output: Contribution to journalArticle

    20 Citations (Scopus)

    Abstract

    Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive L-amino acids to DL-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of D-amino acids, especially D-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. L-Trp (20 mM) was successfully racemized, and the proportion of D-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial L-Trp.

    Original languageEnglish
    Pages (from-to)1299-1305
    Number of pages7
    JournalApplied Microbiology and Biotechnology
    Volume73
    Issue number6
    DOIs
    Publication statusPublished - 2007 Jan

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    Amino Acid Isomerases
    Pseudomonas putida
    Tryptophan
    Amino acids
    Amino Acids
    Substrate Specificity
    Racemases and Epimerases
    Substitution reactions
    Amino Acid Substitution
    Mutagenesis
    Gene encoding
    Substrates
    Screening
    Drug products
    Derivatives
    Catalysts
    Pharmaceutical Preparations
    Genes

    Keywords

    • Broad specificity amino acid racemase
    • D-Tryptophan
    • Random mutagenesis

    ASJC Scopus subject areas

    • Biotechnology
    • Microbiology
    • Bioengineering
    • Microbiology (medical)

    Cite this

    Synthesis of DL-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996. / Kino, Kuniki; Sato, Masaru; Yoneyama, Mariko; Kirimura, Kotaro.

    In: Applied Microbiology and Biotechnology, Vol. 73, No. 6, 01.2007, p. 1299-1305.

    Research output: Contribution to journalArticle

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