Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products

Abstract.

Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as β-D-Fru-(2→1)-β-D-Fru-(2→1)-α-D-glucopyranoside (1-kestose), β-D-Fru-(2→6)-α-D-glucopyranoside (6-β-fructofuranosylglucose), β-D-Fru-(2→1)-β-D-fructofuranoside (inulobiose), β-D-Fru-(2→6)-β-D-Fru-(2→1)-α-D-glucopyranoside (6-kestose) and β-D-Fru-(2→6)-α-D-Glc-(1→2)-β-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.