Purification, characterization, and gene identification of an α-glucosyl transfer enzyme, a novel type α-glucosidase from Xanthomonas campestris WU-9701

Toshiyuki Sato, Nobukazu Hasegawa, Jun Saito, Satoru Umezawa, Yuki Honda, Kuniki Kino, Kotaro Kirimura

    Research output: Contribution to journalArticle

    14 Citations (Scopus)

    Abstract

    The α-glucosyl transfer enzyme (XgtA), a novel type α-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SDS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for α-glucosyl transfer and maltose-hydrolyzing activities using maltose as the α-glucosyl donor, and if necessary, hydroquinone as the acceptor. The V max value for α-glucosyl transfer activity was 1.3 × 10 -2 (mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201, Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13.

    Original languageEnglish
    Pages (from-to)20-27
    Number of pages8
    JournalJournal of Molecular Catalysis B: Enzymatic
    Volume80
    DOIs
    Publication statusPublished - 2012 Aug

    Fingerprint

    Xanthomonas campestris
    Glucosidases
    Maltose
    Purification
    Enzymes
    Genes
    Enzyme kinetics
    Mutagenesis
    Gene encoding
    Glycoside Hydrolases
    Sugar (sucrose)
    Site-Directed Mutagenesis
    Cell Extracts
    Escherichia coli
    Open Reading Frames
    Gel Chromatography
    Sucrose
    Polyacrylamide Gel Electrophoresis
    Catalytic Domain
    Gels

    Keywords

    • α-Glucosidase
    • Gene cloning and expression
    • Glycoside hydrolase family 13
    • Transglucosylation
    • Xanthomonas campestris

    ASJC Scopus subject areas

    • Biochemistry
    • Bioengineering
    • Catalysis
    • Process Chemistry and Technology

    Cite this

    Purification, characterization, and gene identification of an α-glucosyl transfer enzyme, a novel type α-glucosidase from Xanthomonas campestris WU-9701. / Sato, Toshiyuki; Hasegawa, Nobukazu; Saito, Jun; Umezawa, Satoru; Honda, Yuki; Kino, Kuniki; Kirimura, Kotaro.

    In: Journal of Molecular Catalysis B: Enzymatic, Vol. 80, 08.2012, p. 20-27.

    Research output: Contribution to journalArticle

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    abstract = "The α-glucosyl transfer enzyme (XgtA), a novel type α-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SDS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for α-glucosyl transfer and maltose-hydrolyzing activities using maltose as the α-glucosyl donor, and if necessary, hydroquinone as the acceptor. The V max value for α-glucosyl transfer activity was 1.3 × 10 -2 (mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201, Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13.",
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