Levansucrase from Microbacterium laevaniformans ATCC 15953 produced in a 3% sucrose medium was purified to homogeneity from cell-free extracts by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The molecular mass of the purified enzyme was 64 kDa as measured by SDS-PAGE. The optimum pH and temperature for the levan formation were 6.0 and 30degreesC, respectively. The levan-forming activity was strongly inhibited by CuSO4 and HgCl2, and moderately inhibited by ZnSO4. The enzyme synthesized a variety of fructosyl oligosaccharides from various saccharides as fructosyl acceptors. Disaccharides were more favorable fructosyl acceptors than monosaccharides. The structure of the transfer product when melibiose was used as an acceptor was determined by enzyme hydrolysis and C-13 NMR spectroscopy. The chemical structure of the resulting fructosyl melibiose was identified as O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofranoside. This result suggests that levansucrase from M. laevaniformans specifically transferred the fructose moiety of sucrose to the C I-OH position of the glucose residue of melibiose. (C) 2003 Elsevier Science Inc. All rights reserved.