Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese Apricot Prunus mume and cDNA Cloning and Secretory Expression of One of the Isozymes in Pichia pastoris

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Author(s)

    • YURIMOTO Hiroya
    • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
    • SAKAI Yasuyoshi
    • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
    • KOMEDA Hidenobu
    • Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University
    • ASANO Yasuhisa
    • Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University

Abstract

<I>Pm</I>HNL, a hydroxynitrile lyase from Japanese apricot ume (<I>Prunus mume</I>) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the <I>Rosaceae</I> family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (<I>Prunus dulcis</I>). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from <I>Pr. dulcis</I>, inverse PCR method was used for cloning of a putative <I>Pm</I>HNL (<I>Pm</I>HNL2) gene from a <I>Pr. mume</I> seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from <I>Pr. serotina</I>, isozyme 2. The recombinant <I>Pichia pastoris</I> transformed with the <I>Pm</I>HNL2 gene secreted an active enzyme in glycosylated form.

Journal

  • Bioscience, Biotechnology, and Biochemistry

    Bioscience, Biotechnology, and Biochemistry 75(2), 214-220, 2011-02-23

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

References:  33

Cited by:  1

Codes

  • NII Article ID (NAID)
    10027897395
  • NII NACSIS-CAT ID (NCID)
    AA10824164
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    09168451
  • NDL Article ID
    10993100
  • NDL Source Classification
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL Call No.
    Z53-G223
  • Data Source
    CJP  CJPref  NDL  J-STAGE 
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