Synthesis of novel fructooligosaccharides by substrate and enzyme engineering.

Fructooligosaccharides (FOSs) and polyfructosides (PSs) have received particular attention due to its beneficial effects as prebiotics. Here we report the synthesis of a new class of fructooligosaccharides by substrate and enzyme engineering. Using an engineered levansucrase enzyme (SacB of Bacillus subtilis), and sucrose analogues (alpha-Xyl-1,2-beta-Fru or alpha-Gal-1,2-beta-Fru), the product profile shifted from the fructan (levan) polymer to a range of new higher oligosaccharides (xylooligofructosides), or polysaccharides (galactopolyfructosides), of varying size. Further the enzyme was tailored by random mutagenesis, for the synthesis of short-chain fructooligosaccharides to yield variant A5 (N242H), which is unable to produce polymers. It shifts its product pattern to short-chain oligosaccharides and hydrolysis and enabled in combination with the sucrose analogue Xyl-Fru for the first time the direct synthesis of a 6-kestose analogue (alpha-Xyl-1,2-beta-Fru-2,6-beta-Fru). The different glycopyranosyl-residues (i.e. galactose and xylose) that cap fructooligosaccharides may alter prebiotic and biochemical properties.

Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase).

In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in (4)C(1) conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the beta-(1-->2)-fructosyl transfer to 2-OH of L-sugars (L-glucose, L-rhamnose, L-galactose, L-fucose, L-xylose) presumably in a (1)C(4) conformation. In those conformations, the L-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.