Purification And Properties Of The Enzymes Related To The Production Of Fructooligosaccharides From Aspergillus Neger SIPI－602

Purification and properties of the enzymes related to the production of fructooligosaccharides from Aspergillus niger SIPI-602ABSTRACT:Fructooligosaccharides(FOS) are a class of natural oligosaccharides existing widely in many kinds of higher plants and microorganisms. They are mainly composed of l-kestose(GF2), nystose(GF3), 1F- 3 -fructofuranosyl nystose (GF4) in which two, three, and four fructosyl units are bound at the $ -2,1 position of sucrose(GF),respectively.Because of their specific physiological characteristics, i.e., low caloric, non-digestible, noncariogenic, selectively stimulating bifidobacteria growth, antidiabete, FOS have become a class of important health food and alternative sweetener. FOS can be obtained by enzymatic action of a fungal enzyme system, fructosyl transferase on sucrose.Previously, we isolated a FOS-producing fungus, Aspergillus niger SIPI-602. The products converted by Aspergillus niger SIPI-602 mycelia were purified and a pure trisaccharide and a pure tetrasaccharide were obtained. The HPLC chromatograms of both the trimer and tetramer showed single peak and their retention times were identical with GF2 and GF3, respectively. In addition, the structures of the trisaccharide and tetrasaccharide were determined by 13C-NMR and MS.Then, we purified two enzymes related to the FOS production from A.niger SIPI-602, in which P -fructosyltransferase (FTase) converted sucrose into fructooligosaccharides, while ?> -fructofuranosidase hydrolyzes sucrose and fructooligosaccharides into fructose and glucose.The purification protocol consisted of the following procedures: ball milling, ammonium sulfate precipitation and hydrophobic, anion-exchange and gel filtration chromatographies. Both the two enzymes showed single band on SDS-PAGE and the molecular weights of FTase and FFase were 98.86KD and 84.92KD by SDS-PAGE, respectively.The properties of FTase and FFase were also investigated. The Km and Vmax values of FTase with sucrose as a substrate were 0.78mol/L and 4.30mmol/min/L by using the Lineweaver-Buek plot, respectively and the Km and Vmax values of FTase using GFi as a substrate were 0.34mol/L and 0.20mmol/min/L. Glucose was acompetitive inhibitor. FTase was most active at pH4.5-6.0 and at 55"C and stable up to 50 at pH5.8 for one hour of incubation and from pH6.0 to 8.0 at 40癈 in one hour of incubation. The time-course of the formation of FOS from sucrose indicated that the reaction rates increased at a high initial sucrose concentration and the fructose concentration increased at a low sucrose concentration.The Km and Vmax values of FFase for hydrolyzing sucrose into glucose and fructose were 0.025mol/L and 1.39rnmmol/rnin/L respectively. Glucose didn't affect FFase activation and fructose was a competitive inhibitor for FFase. FFase was more active and more stable at acidic conditions. The optimal temperature of FFase was 40癈 and the enzyme was stable below 65癈. The time-course for mycelia, FTase and FFase mixture , and pure FTase were compared respectively and it was confirmed that FFase would slow the time-course of FTase and decrease the FOS concentration in the final product. The results also indicated FOS could induce FFase increase in the reaction course catalyzed by mycelia.