The role of conserved inulosucrase residues in the reaction and product specificity of Lactobacillus reuteri inulosucrase

The probiotic bacterium Lactobacillus reuteri 121 produces two fructosyltransferase enzymes, a levansucrase and an inulosucrase. Although these two fructosyltransferase enzymes share high sequence similarity, they differ significantly in the type and size distribution of fructooligosaccharide products synthesized from sucrose, and in their activity levels. In order to examine the contribution of specific amino acids to such differences, 15 single and four multiple inulosucrase mutants were designed that affected residues that are conserved in inulosucrase enzymes, but not in levansucrase enzymes. The effects of the mutations were interpreted using the 3D structures of Bacillus subtilis levansucrase (SacB) and Lactobacillus johnsonii inulosucrase (InuJ). The wild-type inulosucrase synthesizes mostly fructooligosaccharides up to a degree of polymerization of 15 and relatively low amounts of inulin polymer. In contrast, wild-type levansucrase produces mainly levan polymer and fructooligosaccharides with a degree of polymerization <5. Although most of the inulosucrase mutants in this study behaved similarly to the wild-type enzyme, the mutation G416E, at the rim of the active site pocket in loop 415-423, increased the hydrolytic activity twofold, without significantly changing the transglycosylation activity. The septuple mutant GM4 (T413K, K415R, G416E, A425P, S442N, W486L, P516L), which included two residues from the above-mentioned loop 415-423, synthesized 1-kestose only, but at low efficiency. Mutation A538S, located behind the general acid/base, increased the enzyme activity two to threefold. Mutation N543S, located adjacent to the +1/+2 sub-site residue R544, resulted in synthesis of not such a wide variety of fructooligosaccharides than the wild-type enzyme. The present study demonstrates that the product specificity of inulosucrase is easily altered by protein engineering, obtaining inulosucrase variants with higher transglycosylation specificity, higher catalytic rates and different fructooligosaccharide size distributions, without changing the beta(2-1) linkage type in the product.