title: Structural characterization of bioengineered alpha-D-glucans produced by mutant glucansucrase GTF180 enzymes of Lactobacillus reuteri strain 180.
abstract: Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith degradation analysis, and 1D/2D (1)H NMR spectroscopy. Comparison of the results with structural data of the previously described wild type EPS180 and triple mutant mEPS-PNNS revealed a broad variation of structural elements between mEPS molecules. The amount of (alpha1-->3) linkages varied from 14-43%, the amount of (alpha1-->4) linkages (not present in the wild type) from 0-12%, and the amount of (alpha1-->6) linkages from 51-86%. The average molecular weight (M(w)) ranged from 9.4 to 32.3 MDa and the degree of branching varied from 8-20%. Using a previously established (1)H NMR structural-reporter-group concept, composite models, that include all identified structural features, were formulated for all mEPS molecules. Variations in the mEPS structures strongly affected the physical properties of the mEPSs.
authors.affiliation: Department of Bio-Organic Chemistry, Bijvoet Center, Utrecht University, Utrecht, The Netherlands.
authors.forename: Sander S
authors.initials: SS
authors.lastname: van Leeuwen
authors.forename: Slavko
authors.initials: S
authors.lastname: Kralj
authors.forename: Wieger
authors.initials: W
authors.lastname: Eeuwema
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Gerwig
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
authors.forename: Johannis P
authors.initials: JP
authors.lastname: Kamerling
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D013395:Sucrose / Q000737:chemistry
chemical_list: D011135:Polysaccharides, Bacterial; D013395:Sucrose; D016695:Glycosyltransferases; C033837:alternansucrase