Purification and Characterization of the Enzymes of Fructan Biosynthesis in Tubers of Helianthus tuberosus Colombia (II. Purification of Sucrose:Sucrose 1-Fructosyltransferase and Reconstitution of Fructan Synthesis in Vitro with Purified Sucrose:Sucrose 1-Fructosyltransferase and Fructan:Fructan 1-Fructosyltransferase)

Abstract

Sucrosesucrose 1 -fructosyltransferase (1 -SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianfbus fuberosus that were harvested in the accumulation phase. Cel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27-and a 55-kD fragment. 1 -SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1 -kestose (CF,). Other reactions catalyzed by 1 -SST at a lower rate were self-transfructosylations with CF, and 1,l -nystose (CF,) as substrates yielding CF, and 1,1,1 -fructosylnystose, respectively, as products. 1 -SST also catalyzed the removal of the terminal fructosyl unit from both CF, and CF,, which resulted in the release of sucrose and CF,, respectively, and free Fru. The purified enzyme did not display P-fructosidase activity. An enzyme mixture of purified 1 -SST and fructan:fructan 1 -fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.

Transgenic potato tubers accumulate high levels of 1‐kestose and nystose: functional identification of a sucrose sucrose 1‐fructosyltransferase of artichoke (Cynara scolymus) blossom discs

“…A stimulating effect of pyridoxaI-HCI on fructosyltransferases was also observed by other authors (Koops and Jonker, 1996;Simmen etal., 1993;Wagner and Wiemken, 1986).…”

Isolation of sucrose: sucrose 1‐fructosyltransferase (1‐SST) from barley (Hordeum vulgare)

“…As the sole substrate, 1-kestose cannot be used by the 6-SFT, but is the preferred substrate of the 1-FFT, producing sucrose, nystose (1,1 kestotetraose) and higher DP inulins. Incubation of 1-kestose with purified 1-SST from Asteraceae yielded small amounts of nystose and significant amounts of sucrose and fructose (Koops & Jonker, 1996 ;Lu$ scher et al, 1996 ;Van den Ende et al, 1996). Therefore, analysis of product formation combined with the F : G ratio clearly distinguishes 1-SST from 6-SFT and FFT activities.…”

“…Incubation of the first 1-SST form (sections 8-10) with 1-kestose showed that the enzyme also acts as an exohydrolase (Figs 1b, 2), producing fructose as described for the 1-SSTs purified from Asteraceae (Koops & Jonker, 1996 ;Lu$ scher et al, 1996 ;Van den Ende et al, 1996). The sucrose generated is then used as a substrate by the 1-SST to produce 1-kestose, as indicated by the glucose production in the assays (Fig.…”

“…2.4.1.100) that transfers fructose moieties between fructan molecules (Koops & Jonker, 1996 ;Lu$ scher et al, 1996 ;Van den Ende & Van Laere, 1996). Liliaceae have similar fructans but with a fructosyl chain also attached to the 6-position of the glucosyl residue (inulin neoseries).…”

“…The enzyme 1-SST has been purified and characterized from several plants producing inulins or inulin neoseries from the families of the Asteraceae and Liliaceae (Shiomi & Izawa, 1980 ;Koops & Jonker, 1996 ;Lu$ scher et al, 1996 ;Van den Ende et al, 1996), and corresponding cDNAs have recently been cloned (Hellwege et al, 1997 ;Van der Meer et al, 1998 ;Vijn et al, 1998) ; sequence information revealed a similarly close relationship between 1-SST of these plants and invertase as in the case of 6-SFT Moreover, 1-SST is also obviously important in the synthesis of 2,6-linked graminans of grasses, and SST activity has been extensively studied in barley (reviewed by Wiemken et al, 1995). However, 1-SST has never been purified from barley or any other grass, except an enzyme with unusually broad activity from tall fescue (Lu$ scher & Nelson, 1995).…”