Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10⁵ times reduction of total sucrase activity.     

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935-941] has provided evidence for the presence of a bound metal ion, most likely Ca2+. Characterization of site-directed mutants in the putative Ca2+ ion-binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca2+ binding. Sequence alignments of family GH68 proteins showed that this Ca2+ ion-binding site is (largely) present only in proteins of Gram-positive origin.     

Inulin and its enzymatic production by inulosucrase: Characteristics,structural features,molecular modifications and applications

Inulin, a natural fructan, cannot be hydrolyzed by digestive enzymes in the human body and plays a role as a dietary fiber and prebiotic. Due to its versatile physicochemical properties and physiological functions, inulin has been widely applied in food, pharmaceuticals, and many other fields. The microorganism-derived inulin-forming enzyme inulosucrase (ISase) (EC: 2.1.4.9) can biosynthesize higher-molecular-weight inulin than plants using sucrose as the sole substrate, and the enzyme also shows transfructosylation activity toward other saccharide acceptors. In this article, the properties, functions, and applications of inulin are overviewed. The biosynthesis of inulin by ISase is addressed, including ISase characteristics, structural features, molecular modifications and applications.     

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum

Background

IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme.

Results

Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS.

Conclusions

Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.     

Biochemical properties of inulosucrase from Leuconostoc citreum CW28 used for inulin synthesis

Biochemical properties of inulosucrase from Leuconostoc citreum CW28, a potential biocatalyst for inulin synthesis, were determined in order to select optimal reaction conditions. The hydrolysis reaction was about 3.5 times more efficient than the transferase reaction. It was found that high sucrose concentrations (≈250 g L^-1) were required for maximum fructose transferase yields. High molecular weight inulin distributions were obtained with cell associated inulosucrase, while lower size products were associated to the activity of the free enzyme in solution. When using whole cells, mannitol was found as a by-product of the reaction resulting from the reduction of fructose released by sucrose hydrolysis. A 30 L pilot plant synthesis with 250 g L^-1 of sucrose was carried out using the cell associated inulosucrase resulting in 76% of the substrate being transformed to inulin.