Structural analysis of bioengineered alpha-D-glucan produced by a triple mutant of the Glucansucrase GTF180 enzyme from Lactobacillus reuteri strain 180: generation of (alpha1-->4) linkages in a native (1-->3)(1-->6)-alpha-D-glucan.
Site-directed mutagenesis of the glucansucrase gtf180 gene from Lactobacillus reuteri strain 180 was used to transform the active site region. The alpha-D-glucan ( mEPS-PNNS) produced by the triple mutant V1027P:S1137N:A1139S differed in structure from that of the wild-type alpha-D-glucan ( EPS180). Besides (alpha1-->3) and (alpha1-->6) linkages, as present in EPS180, mEPS-PNNS also contained (alpha1-->4) linkages. Linkage analysis, periodate oxidation, and 1D/2D (1)H NMR spectroscopy of the intact mEPS-PNNS, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of mEPS-PNNS afforded a composite model, which includes all identified structural features.
van Leeuwen
Sander S
SS
Department of Bio-Organic Chemistry, Bijvoet Center, Utrecht University, 3584 CH Utrecht, The Netherlands.
Kralj
Slavko
S
Gerwig
Gerrit J
GJ
Dijkhuizen
Lubbert
L
Kamerling
Johannis P
JP
D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods; D005936:Glucans; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019344:Lactic Acid / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010504:Periodic Acid / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods
D000838:Anions; D005936:Glucans; D010504:Periodic Acid; D019344:Lactic Acid; C009288:metaperiodate; D016695:Glycosyltransferases; C033837:alternansucrase; D010100:Oxygen