The Purification And Properties Of An Intracellular ~6 G- Fructofuranosidase From Xanthophyllomyces Dendrorhous

Using sucrose as the substrate, ⁶G-fructofuranosidases from Xanthophyllomyces dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Neokestose withβ-2, 6 linkage between fructose and a glucosyl group (⁶G-FOSs) has better prebiotic properties and chemical stability compared to FOSs.In this paper, FOSs produced by X. dendrorhous 269 as the starting strain was studied. Three kinds of wall breaking methods were used to release the intracellular ⁶G-fructofuranosidase from Xanthophyllomyces dendrorhous, the results indicated that the wall-broken rate on cell disruption system was 89.8%, 87.5% for high-pressure homogenizing system, which all significantly better than that 50.1% by repetitive freeze thaw method.The enzyme was purified by DEAE-52 cellulose chromatography. Thirty-five-fold purification, a 13.4% enzyme activity recovery and a specific activity of 238.64U/mg were achieved. The purified enzyme was a single band of around 33 kDa on SDS-PAGE.Finally, properties of the intracellular ⁶G-fructofuranosidase were studied. Optimum enzyme activity occurred at pH 6.4 and 45°C and the enzyme was stable at pH 4.0-7.0 and at 45°C. K⁺ and Mg²⁺ had no obvious effect on the activity of endo-⁶G-FFase. However, Ca²⁺, Li⁺, Mn²⁺, Ba²⁺ and Al³⁺ caused a 1.25?1.4 fold increase and a slight inhibition was induced by Zn²⁺. EDTA increased the enzyme activity whereas SDS significantly reduced its activity. Using sucrose as a substrate, the Km and Vmax values were, respectively, 511 mmol/l and 233μmol/(min·mg) for transfer activity and 62 mmol/l and 164μmol/(min·mg) for hydrolytic activity. Under optimum conditions, a maximum concentration (73.9 g/l) of neo-fructooligosaccharides catalyzed by the endo-enzyme was obtained.The crude enzyme was widely used in industry, the characterization of the crude intracellular ⁶G-fructofuranosidase was studied, and the results indicated that the optimum enzyme activity was at pH 6.4 and 60°C and the enzyme was stable at pH 4.0-7.0 and below 70°C. A slight inhibition was induced by Zn²⁺ and Mg²⁺, and SDS significantly reduced the activity. It indicated 64.56% of relative activity. Ba²⁺ and Al³⁺ had a slight increase on the enzyme. Other metal ions and reagents had no obvious effect on the activity of endo-enzyme.