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:
Km 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 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.