Purification and Characterization of an Oat Fructan Exohydrolase That Preferentially Hydrolyzes P-2,6-Fructans1

Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of /3-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal p-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal p-2,6 linkage of 6G-kestotriose and approximately 1 O times faster than it hydrolyzed the terminal p-2,1 linkage of chicory inulin. Sucrose and I-kestose were not substrates. The K, for neokestin (p-2,6-linked fructans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v), and the V,,, was 0.041 pmol min-’ mL-’. The K,,, for hydrolysis of 6C,6-kestotetraose was 5.6% (w/v), and the V,,, was 0.1 38 pmol min-’ mL-’. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.