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, Kohtaro Kirimura

研究成果: Article

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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.

元の言語English
ページ(範囲)20-27
ページ数8
ジャーナルJournal of Molecular Catalysis B: Enzymatic
80
DOI
出版物ステータスPublished - 2012 8 1

ASJC Scopus subject areas

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology

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