• Toxicological Research
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• v.20 no.3
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• pp.263-272
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• 2004

This study was to investigate single and repeated-dose toxicities of DFA IV, a new candidate of nutraceutical which has preventive effect on anemia and osteoporosis. In single-dose oral toxicity study, the test article were administered once by gavage to rats at dose level of 0, 2,000 and 5,000 mg/kg. No dead animal, abnormal sign and abnormal necropsy finding was found in control and treated groups. Thus the approximate lethal dose of DFA IV was considered to be higher than 5,000 mg/kg in rats. In four week repeated dose oral toxicity study, the test article was administered once daily by gavage to rats at dose levels of 0, 500, 1,000 and 2,000 mg/kg. No abnormality was observed in mortality, clinical findings, body weight changes, food and water consumptions, opthalmoscopic findings, hematological findings, necropsy findings, organ weights and histopathological findings. In urinalysis, specific gravity was increased in 2,000 mg/kg groups of male rats. In serum biochemical analysis, creatine phosphokinase was increased in all treatment groups of male rats. These increases in urine specific gravity and serum creatine phosphokinase activity were not accompanied with related signs such as histopathological changes or clinical findings. In conclusion, four week repeated oral dose of DFA IV to rats did not cause apparent toxicological change at the dose of 500, 1,000 or 2000 mg/kg body weight. Thus it is suggested that no-observed-adverse-effect level (NOAEL) of DFA IV in rats would be 2,000 mg/kg/day body weight.

• 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.