• KOREAN JOURNAL OF CROP SCIENCE
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• v.41 no.4
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• pp.456-464
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• 1996

Fructans are the major vegetative storage carbohydrate in wheat(Triticum aestivum L.). The depolymerization of fructans occurs by the sequential removal of terminal fructosyl residues by a specific fructan exohydrolase(FEH). The objective of this study was to isolate and characterize this enzyme in wheat. From stems and sheaths of field-grown wheat(cv. Clark), FEH was purified 356-fold using salt precipitation and a series of chromatographic procedures including size exclusion, anion exchange, and affinity chromatography. FEH had a molecular weight of 63.7 kD and an optima at pH 5.5 and 3$0^{\circ}C$. The $K_{m}$s for $\beta$(2 longrightarrow1) linked oligofructans varied, from 10 to 37mM, with the lowest $K_{m}$ for tetrasaccharide. The $V_{max}$ increased as degree of polymerization (DP) increased. Wheat FEH hydrolyzed only, $\beta$(2 longrightarrow1) linked fructans but not, $\beta$(2 longrightarrow6) linked timothy fructan or sucrose. The role of this FEH in fructan metabolism in wheat is discussed.sed.

• Journal of Life Science
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• v.19 no.9
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• pp.1218-1225
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• 2009

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at $55^{\circ}C$. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less $\alpha$-helix and more $\beta$ strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.