Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus

Abstract

A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% w/w carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60ºC. It was stable or slightly activated (up to 1.4-fold) in the presence of reducing agents such as dithiotreitol and 2-mercaptoethanol, and detergents such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the kcat and Km values for transfructosylating activity were 1.62  0.09 104 s-1 and 0.53  0.05 M, whereas for hydrolytic activity the corresponding values were 775  25 s-1 and 27  3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.