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Purification and characterization of 1‐SST, the key enzyme initiating fructan biosynthesis in young chicory roots ( Cichorium intybus )
Author(s) -
Ende Wim,
Wonterghem Dominik,
Dewil Erna,
Verhaert Peter,
Loof Arnold,
Laere André
Publication year - 1996
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1996.tb05699.x
Subject(s) - cichorium , fructan , invertase , biochemistry , ammonium sulfate precipitation , sucrose , enzyme , enzyme assay , molecular mass , chemistry , biology , size exclusion chromatography , chromatography , botany
A genuine 1‐SST (sucrose:sucrose 1‐fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63‐fold purification and a specific activity of 4.75 U (mg protein) −1 . The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS‐PAGE apparent molecular masses of 49 kDa (α‐subunit) and 24 kDa (β‐subunit) were found. Further specification was obtained by MALDI‐TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS‐boiled chicory root extract and chicken‐raised polyclonal antibodies against the purified 1‐SST, indicating that the enzyme is a heterodimer in vivo. The N‐terminus of chicory root 1‐SST α‐subunit was shown to be highly homologous with the cDNA‐derived amino acid sequences from barley 6‐SFT and a number of β‐fructosyl hydrolases (in‐vertases and fructan hydrolases). However, chicory root 1‐SST properties could be clearly differentiated from those of chicory root 1‐FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1‐kes‐tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1‐SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1‐FFT and barley 6‐SFT, the enzyme also showed some β‐fructofuranosi‐dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β‐fructofuranosidase activity were also detected using 1‐kestose as the sole substrate.