SEASONAL-VARIATION OF FRUCTAN-BETA-FRUCTOSIDASE (FEH) ACTIVITY AND CHARACTERIZATION OF A BETA-(2-1)-LINKAGE SPECIFIC FEH FROM TUBERS OF JERUSALEM-ARTICHOKE (HELIANTHUS-TUBEROSUS)

The fructan-beta-fructosidase activity (1-FEH; EC 3.2.1.80) that degra des inulin in tubers of Helianthus tuberosus L. appears to be developm entally regulated; it was low in growing tubers but increased during d ormancy and sprouting. In spite of relatively high 1-FEH activity in v ine, fructose concentration was very low in developing and dormant tub ers and increased markedly only during sprouting. A fructan-beta-fruct osidase from such sprouting tubers was purified drl-fold to a single p rotein band on one-dimensional sodium dodecylsulphate-polyacrylamide g els. The purification procedure included ammonium sulphate precipitati on, lectin-affinity chromatography on concanavalin A, anion-exchange a nd cation-exchange chromatography. The enzyme had an apparent molecula r mass of 75000 measured by size-exclusion chromatography, and 79000 m easured by one-dimensional sodium dodecylsulphate-polyacrylamide gel e lectrophoresis. It exhibited a high substrate specificity, hydrolysing terminal beta-(2-1)-fructosyl-fructose-linkages in linear and branche d fructan oligomers; beta-(2-6)-linkages were hardly hydrolysed. Hydro lysis of inulin oligomers followed normal saturation kinetics: K-m val ues for 1,1-kestotetraose and 1,1,1-kestopentaose were 8.3 mM and 12 m M, respectively. Fructosyl residues were hydrolysed from inulin oligom ers by a multi-chain mechanism. The fructan-beta-fructosidase showed o ptimal enzyme activity at pH 5.2, and it retained its full activity af ter pre-incubation for 1 h at up to 40 degrees C. The release of fruct ose from 5 mM 1,1-kestotetraose was reduced by 25 % when 1-FEH was ass ayed in the presence of 10 mM sucrose. It is proposed that the inhibit ion of 1-FEH activity by sucrose is a mechanism for controlling fructa n degradation in planta.