Seasonal variation of fructan‐β‐fructosidase (FEH) activity and characterization of a β‐(2‐1)‐linkage specific FEH from tubers of Jerusalem artichoke ( Helianthus tuberosus )

SUMMARY The fructan‐β‐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 vitro , fructose concentration was very low in developing and dormant tubers and increased markedly only during sprouting. A fructan‐β‐fructosidase from such sprouting tubers was purified 41‐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 β‐(2‐1)‐fructosyl‐fructose‐linkages in linear and branched fructan oligomers; β‐(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‐β‐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°C. The release of fructose from 5 m m 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 .