Structural characterization of mixed-linkage α-glucans produced by mutants of Lactobacillus reuteri TMW 1.106 dextransucrase.

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.

Detailed Structural Characterization of Glucans Produced by Glucansucrases from Leuconostoc citreum TMW 2.1194.

The water kefir organism Leuconostoc citreum TMW 2.1194 forms highly branched dextrans with O3- and O4-bound side chains. To obtain detailed information on the enzymatic synthesis of these polymers, the four glucansucrases encoded by Leuconostoc citreum TMW 2.1194 were cloned, heterologously expressed, and used for polysaccharide production. Molecular and macromolecular structure of the synthesized glucans were analyzed by methylation analysis, two-dimensional NMR spectroscopy, oligosaccharide analysis after partial hydrolysis, and asymmetric flow field-flow fractionation. It was demonstrated that two glucansucrases form insoluble glucans with variously branched dextran sections and varying portions of consecutive, 1,3-linked glucose units. In contrast, the other two glucansucrases synthesized O3- (Lc6255) and O4-branched (Lc1785) soluble dextrans. Analysis, isolation, and characterization of enzymatically liberated oligosaccharides showed that monomeric and elongated side chains are abundant in both polysaccharides. From the structures and size distributions it was concluded that Lc1785 is mainly responsible for synthesis of fermentatively produced soluble dextrans.

Fine structures of different dextrans assessed by isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides.

Chromatographic analysis of endo-dextranase liberated branched oligosaccharides proved to be a valuable approach to differentiate dextrans from fermented food products or isolated lactic acid bacteria. Because these hydrolysis products also yield valuable information on the dextran fine structures, several branched isomalto-oligosaccharides were liberated from different dextrans and chromatographically purified. Mass spectrometry and two-dimensional NMR spectroscopy were used for structural characterization of the oligomers. Isomalto-oligosaccharides from L. reuteri TMW 1.106 dextrans were exclusively O4-branched. Furthermore, they contained only monomeric side chains and at least one unsubstituted backbone unit between ramified residues. In contrast, O3-branched oligosaccharides with monomeric as well as elongated side chains were isolated. The varying abundance of the O3-branched oligosaccharides suggests that side chain length is a major factor for structural differences between dextrans. Overall, we demonstrated that chromatographic analysis of endo-dextranase liberated isomalto-oligosaccharides provides valuable information on the structural properties and supplements conventional methods such as methylation analysis.