Characterization of a fructan exohydrolase purified from barley stems that hydrolyzes multiple fructofuranosidic linkages

Barley (Hordeum vulgare cv Morex) fructan exohydrolase (EC 3.2.1.80) was purified by precipitation with ammonium sulfate and chromatography on anion exchange and lectin affinity columns. The final enzyme preparation was homogenous as determined by the presence of a single band on silver stained SDS-PAGE and IEF gels. The purified protein had a molecular mass of 33 kDa and a pI of 7.8. Analyses of relative hydrolytic rates of various fructans were determined by measuring released fructose by pulsed electrochemical detection after separation of reactions by HPLC. The purified enzyme hydrolyzed β-2,1-linkages in 6G, 1-kestotetraose, 1 and 6G-kestotetraose, 1, 1-kestotetraose, and 1-kestotriose with relative rates of 100 : 96 : 85 : 88. This enzyme slowly hydrolyzed the β-2,6-linkages in 6G-kestotriose and in 6G, 6-kestotetraose and sucrose with relative rates of 5 : 4 : 3 compared to 6G, 1-kestotetraose hydrolysis rates arbitrarily set at 100. The substrate attack pattern, determined by identifying products from hydrolysis of purified fructan tetrasaccharides, was of the multichain type. Sucrose was a mixed-type inhibitor of inulin hydrolysis.