Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris
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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.
KeywordsGlucosamine-6-phosphate synthase Pichia pastoris Saccharomyces cerevisiae
This work was financially supported by the National High-tech R&D Program of China (863 Program, No. 2012AA021504) and the Taishan Scholar Program of Shandong.