Enzymatic synthesis of fructooligosaccharides by levansucrase from Bacillus amyloliquefaciens: specificity, kinetics, and product characterization

The kinetic parameters and the product spectra of purified Bacillus amyloliquefaciens levansucrase (LS) were investigated in the present study. LS was highly transfructosylic than hydrolytic with high kcat value of 1136.5 s−1 for its transfructosylation activity. Contrary to the hydrolytic activity, the kinetic of the transfructosylation activity of LS was best fitted to the Hill model with low Hill coefficients (nH, 0.74), indicating the negative binding cooperativity of substrates. These results reveal the competition of sucrose with the growing (oligo)polymer chain as a fructosyl acceptor. A shift of the transfructosylation reaction further towards the polymerization side was observed in the presence of raffinose as a fructosyl donor as compared to sucrose. The acceptor specificity studies reveal the ability of LS to synthesize a variety of hetero-fructooligosaccharides from various saccharides as fructosyl acceptors. Disaccharides were more favourable fructosyl acceptors as compared to monsaccharides. Contrary to disaccharides acceptor reactions, LS-catalyzed transfructosylation of monosaccharides did not yield a quasi equilibrium state. Contrary to lactose, galactose, xylose and glucose acceptor reactions, a lower accumulation of levan was detected in the presence of maltose. When sucrose and raffinose coexisted, they were both used as fructosyl donor, revealing the low acceptor specificity of LS towards raffinose. The structures of products were analysed NMR and MS.

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► B. amyloliquefaciens levansucrase (LS) has one of the highest turnover numbers for the catalysis of the transfructosylation reaction.
► A shift of the transfructosylation reaction further towards the polymerization side was observed in the presence of raffinose donor as compared to sucrose.
► LS synthesized a variety of hetero-fructooligosaccharides from various fructosyl acceptors with disaccharides being the best ones.
► The formation of polymer fructans (Gal-(Fru)n-Fru, n > 18) was the most abundant in the galactose-acceptor reaction.
► The oligomers (Xyl-(Fru)n-Fru, n < 18) were the dominant transfructosylation products in the xylose-acceptor reaction.