PURIFICATION AND CHARACTERIZATION OF AN OAT FRUCTAN EXOHYDROLASE THATPREFERENTIALLY HYDROLYZES BETA-2,6-FRUCTANS

Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammoni um sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogene ity as determined by the presence of a single band (43 kD) on a silver -stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of beta- 2,6-linked fructan (neokestin) isolated from oat was used as the subst rate to purify fructan hydrolase. Neokestin and small degree of polyme rization fructan isomers were used to characterize the substrate speci ficity of the purified enzyme. The purified fructan hydrolase catalyze d hydrolysis of the terminal beta-2,6 linkage of 6(G),6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6 linkag e of 6(G)-kestotriose and approximately 10 times faster than it hydrol yzed the terminal beta-2,1 linkage of chicory inulin. Sucrose and 1-ke stose were not substrates. The K-m for neokestin (beta-2,6-linked fruc tans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v ), and the V-max was 0.041 mu mol min(-1) mL(-1). The K-m for hydrolys is of 6(G),6-kestotetraose was 5.6% (w/v), and the V-max was 0.138 mu mol min(-1) mL(-1). Catalysis was exolytic acid by multiple chain atta ck. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.