title: Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli.
abstract: The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the Km and Vmax of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the Km for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction.
authors.affiliation: Wood Research Institute, Kyoto University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nakai
authors.forename: N
authors.initials: N
authors.lastname: Tonouchi
authors.forename: T
authors.initials: T
authors.lastname: Tsuchida
authors.forename: H
authors.initials: H
authors.lastname: Mori
authors.forename: F
authors.initials: F
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
mesh_terms: D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007887:Fabaceae / Q000201:enzymology*; D005964:Glucosyltransferases / Q000032:analysis / Q000096:biosynthesis*; D007700:Kinetics; D010946:Plants, Medicinal; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D005964:Glucosyltransferases; C022699:sucrose synthase