Purification and Biological Characterization of Wild-type and Mutants of a Levan Fructotransferase from Microbacterium sp. AL-210

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFAⅠ, 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 55oC. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less α-helix and more β 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.