Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Biotechnol Lett. 2014 Oct;36(10):2023-8. doi: 10.1007/s10529-014-1561-y. Epub 2014 Jun 15.

Abstract

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml(-1) for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein (-1). The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The Km and Vmax of the GlcN-6-P synthase towards D-fructose 6-phosphate were 2.8 mM and 6.9 μmol min(-1) mg(-1), respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / biosynthesis*
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / chemistry
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / genetics
  • Models, Molecular
  • Pichia / enzymology
  • Pichia / genetics*
  • Protein Structure, Secondary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / biosynthesis*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics

Substances

  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • GFA1 protein, S cerevisiae
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)