Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli

Biosci Biotechnol Biochem. 1997 Sep;61(9):1500-3. doi: 10.1271/bbb.61.1500.

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.

MeSH terms

  • Blotting, Western
  • Chromatography, Gel
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology*
  • Fabaceae / enzymology*
  • Glucosyltransferases / analysis
  • Glucosyltransferases / biosynthesis*
  • Kinetics
  • Plants, Medicinal*
  • Plasmids / genetics
  • Recombinant Proteins / biosynthesis

Substances

  • Recombinant Proteins
  • Glucosyltransferases
  • sucrose synthase