Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli.
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.
Nakai
T
T
Wood Research Institute, Kyoto University, Japan.
Tonouchi
N
N
Tsuchida
T
T
Mori
H
H
Sakai
F
F
Hayashi
T
T
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
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C022699:sucrose synthase