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We previously reported that β-glucosidase BGL1 at low concentration (15 μg mL -1 ) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 μg mL -1 ) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 μg mL -1 ) showed transglycosylation but BGL1 at high concentration (200 μg mL -1 ) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a β(1 → 3) or β(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-β-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF.HS.FGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF or FGFR. In this report, we used a new strategy to study the structural specificity of HS binding to 10 different FGF.FGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desulfated heparin, and HS were used for the interaction studies by solution competition surface plasmon resonance (SPR) and filter trapping assays. Specific oligosaccharides binding to FGF.FGFR complexes were subjected to polyacrylamide gel electrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography and mass spectrometry. The competition SPR studies using sized oligosaccharide mixtures showed that binding of each of the tested FGFs or FGF.FGFR complexes to heparin immobilized to an SPR chip was size-dependent. The 6-desulfated heparin oligosaccharides exhibited a reduced level of inhibition of FGF and FGF.FGFR complex binding to heparin in the competition experiments. Heparin and the 6-desulfated heparin exhibited higher levels of inhibition of the FGF.FGFR complex binding to heparin than of FGF binding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between the FGF.FGFR complexes and oligosaccharides. Disaccharide analysis showed that HS disaccharides with a degree of polymerization of 10 (dp10) had high binding selectivity, while dp10 heparin and dp10 6-desulfated heparin showed reduced or no selectivity for the different FGF.FGFR complexes tested.

Isobaric oligosaccharides enzymatically prepared from hyaluronic acid (HA) and N-acetylheparosan (NAH), were distinguished using tandem mass spectrometry. The only difference between the two series of oligosaccharides was the linkage pattern (in HA 1→3 and in NAH 1→4) between glucuronic acid and N-acetylglucosamine residues. Tandem mass spectrometry afforded spectra in which glycosidic cleavage fragment ions were observed for both HA and NAH oligosaccharides. Cross-ring cleavage ions 0,2An and 0,2An-h (n is even number) were observed only in GlcNAc residues of NAH oligosaccharides. One exception was an 0,2A2 ion fragment observed for the disaccharide from HA. These cross-ring cleavage fragment ions are useful to definitively distinguish HA and NAH oligosaccharides. [Copyright &y& Elsevier]

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with 0,2A- and 2,5A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Zint-ions (Zint − 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [2,5Ared]/[0,4Ared] (red: denotes reducing terminal). [Copyright &y& Elsevier]

Investigation of the action pattern of enzymes acting on carbohydrates is challenging, as both the substrate and the digestion products are complex mixtures. Dextran and its enzyme-derived oligosaccharides are widely used for many industrial applications. In this work, a new method relying on ultra-performance hydrophilic interaction liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UP-HILIC- Q/TOF-MS/MS) was developed to analyze a complex mixture of dextran oligosaccharide products to determine the action pattern of dextranase. No derivatization of oligosaccharides was required and the impact of the α- and β-configurations of the native oligosaccharides on the chromatographic separation was eliminated. The 1→6, 1→3, 1→4 backbone linkages and the branch linkages of these oligosaccharides were all distinguished from diagnostic ions in their MS/MS spectra, including fragments corresponding to A, A, A, B-HO, A, and A. The sequences of the oligosaccharide products were similarly established. Thus, the complex oligosaccharide mixtures in dextran digestion products were profiled and identified using this method. The more enzyme-resistant structures in dextran were established using much less sample, labor, time, and uncertainty than in previous studies. This method provides an efficient, sensitive, and straightforward way to monitor the entire process of digestion, establish the action pattern of the dextranase from Penicillium sp., and to support the proper industrial application of dextranase. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]