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 degrades inulin in tubers of Helianthus tuberosus L. appears to be developmentally regulated; it was low in growing tubers but increased during dormancy and sprouting. In spite of relatively high 1-FEH activity in vine, fructose concentration was very low in developing and dormant tubers and increased markedly only during sprouting. A fructan-beta-fructosidase from such sprouting tubers was purified drl-fold to a single protein band on one-dimensional sodium dodecylsulphate-polyacrylamide gels. The purification procedure included ammonium sulphate precipitation, lectin-affinity chromatography on concanavalin A, anion-exchange and cation-exchange chromatography. The enzyme had an apparent molecular mass of 75000 measured by size-exclusion chromatography, and 79000 measured by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis. It exhibited a high substrate specificity, hydrolysing terminal beta-(2-1)-fructosyl-fructose-linkages in linear and branched fructan oligomers; beta-(2-6)-linkages were hardly hydrolysed. Hydrolysis of inulin oligomers followed normal saturation kinetics: K-m values for 1,1-kestotetraose and 1,1,1-kestopentaose were 8.3 mM and 12 mM, respectively. Fructosyl residues were hydrolysed from inulin oligomers by a multi-chain mechanism. The fructan-beta-fructosidase showed optimal enzyme activity at pH 5.2, and it retained its full activity after pre-incubation for 1 h at up to 40 degrees C. The release of fructose from 5 mM 1,1-kestotetraose was reduced by 25 % when 1-FEH was assayed in the presence of 10 mM sucrose. It is proposed that the inhibition of 1-FEH activity by sucrose is a mechanism for controlling fructan degradation in planta.