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

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.

Expression ofAspergillus nigerglucose oxidase in yeastPichia pastorisSMD1168

“…Through slowing down the proteolysis, the production of target protein will be increased. Several studies have shown that the protease-deficient strain SMD1168 could highly express lipoxygenase, [17] lysozyme LYZL6, [18] glucosamine-6-phosphate synthase, [19] streptokinase [20] and other exogenous proteins.…”