Overproduction, purification, and property analysis of an extracellular recombinant fructosyltransferase

Abstract

Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase Ca2+ and Mg2+ absorption. In this study, the fructosyltransferase (FruSG) from Aspergillus niger strain SG610, which is used in the industrial production of FOS, was successfully overexpressed in Pichia pastoris strain GS115 and characterized. Recombinant FruSG had optimum temperature and pH of 50 °C and 5.5, respectively, and it was stable below 40 °C and at from pH 3.0 to 8.0. Km and kcat values of the recombinant FruSG were 424.5 mmol L−1 and 3.8 × 103 min−1, respectively. On addition of 50 and 100 g L−1 glucose, the Ki was 130.6 and 170.0 mmol L−1, respectively. The recombinant FruSG had the highest activity (2294.7 U mL−1) after induction for 144 h in a 5-L fermenter, which was 956.1-fold higher than that of native FruSG from A. niger SG610, and produced a high yield of FOS (64.5 %, w/w). The recombinant FruSG was activated by 5 mmol L−1 Mg2+ and 1 mmol L−1 Fe2+ (relative activity of 125.2 and 120.9 %, respectively), indicating that the enzyme was Mg2+ and Fe2+ dependent. Moreover, toluene, n-butanol, trichloromethane, and ethyl acetate significantly activated FruSG. Together, these results indicate that the recombinant FruSG is an excellent candidate for the industrial production of FOS.