Β-linked Oligosaccharides Research Articles

Is beer a source of prebiotics?

Author(s): Kanyer, AJ; Bornhorst, GM; Marco, ML; Bamforth, CW | Abstract: Beer contains low-molecular-weight β-linked oligosaccharides that originate from the degradation of β-glucan in the barley cell wall during malting and mashing. Over 90% of these oligosaccharides contain three or four glucosyl units. They remain intact through a static oral, gastric and small intestinal in vitro human digestive system model, indicating that they should be available to beneficial organisms known to be present in the human large intestine. Several intestine-associated Lactobacillus strains were shown to be capable of growth on these β-linked oligosaccharides, thereby leading us to tentatively propose that these compounds may represent prebiotics. Copyright © 2017 The Institute of Brewing a Distilling.

Isolation and characterization of a novel thermostable β-glucosidase from Bacillus subtilis SU40.

A new bacterial strain SU40 with ability to produce thermostable β-glucosidase was isolated from soil located at Puducherry (India) and identified as Bacillus subtilis. The conditions like temperature, pH, carbon and nitrogen sources were optimized conditions for the maximum production of β-glucosidase. The enzyme was purified using ammonium sulphate precipitation and BioSep and HPLC chromatography. Purified enzyme was found to be a monomeric protein with an apparent molecular mass of 38.3 kDa. The enzyme was stable over a wide range of pH and temperatures and exhibited maximal activity at 75°C and pH 12.0. The enzyme was found to retain approximately 60% of maximal activity for 1 h at 70°C and hydrolyzed a wide range of aryl-β-D-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and p-nitrophenyl-β-D-glucopyranoside. The Km and Vmax values of the enzyme were 0.21 μM and 9.38 μmoles mg(-1) min(-1), respectively. The enzyme demonstrated stability in the presence of 0.5% surfactants and was completely inhibited after addition of EDTA and Hg2+ ions. Synergistic action of β-glucosidase from B. subtilis SU40 with endoglucanase from Paenibacillus barcinonensis MG7 completely degraded the rice straw.

Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities

BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

Glycal assembly by the in situ generation of glycosyl dithiocarbamates.

Glycal assembly offers an expedient entry into β-linked oligosaccharides, but epoxyglycal donors can be capricious in their reactivities. Treatment with Et(2)NH and CS(2) enables their in situ conversion into glycosyl dithiocarbamates, which can be activated by copper triflate for coupling with complex or sterically congested acceptors. The coupling efficiency can be further enhanced by in situ benzoylation, as illustrated in an 11-step synthesis of a branched hexasaccharide from glucals in 28% isolated yield and just four chromatographic purifications.

A novel two-step synthesis of α-linked mannobioses based on an acid-assisted reverse hydrolysis reaction

Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides, we propose here a versatile new approach. By Fischer type glycosylation, a d-mannose solution of extremely high concentration (approximately 83% (w/w)) was incubated at 60 °C for 65 h in 0.5 M HCl. After dilution and neutralization, the small amount of formed β-linked oligosaccharides was hydrolyzed by β-mannosidase. The yields of α- d-Man p-(1→2)- d-Man p (7.9%), α- d-Man p-(1→3)- d-Man p (7.9%), and α- d-Man p-(1→6)- d-Man p (29.1%) isolated by an activated carbon column chromatography were almost identical to those of the enzymatic reaction, but the yield of α- d-Man p-(1→3)- d-Man p increased enormously by the present method.

Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus

The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg + Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30 °C and it exhibited maximal activity at 25 °C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β- d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K m and k cat were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium.

A cell wall-bound β-Glucosidase from germinated rice: Purification and properties

A cell wall-bound β-glucosidase was purified from germinated rice seeds, and its N-terminal amino acid sequence was determined. The enzyme had a molecularmass of 56 kDa and an isoelectric point of > 10.0. The enzyme showed hydrolytic as well as trans-glycosylation activity towards β-linked oligosaccharide substrates, suggesting that it may be involved in hydrolysis as well as reorganization of oligosaccharides in cell walls during germination. A large portion of β-glucosidase (EC 3.2.1.21) in germinating rice seeds, which appears to be ionically bound to cell walls, can be solubilized with 1 M NaCl. Its activity increased more than eight-fold within five days of germination. It was purified to electrophoretic homogeneity from the extracts of germinated rice seeds by fractionation with (NH 4) 2SO 4 followed by CM-Sepharose, Polybuffer exchanger 118, Concanavalin A-Sepharose and Bio-Gel P-100. The Mr of the purified enzyme, estimated by SDS-PAGE, was 56,000 and the isoelectric point was > 10.0. Its N-terminal amino acid sequence (44 residues) exhibited high homology to those of β-glucosidases from other plants, such as barley and white clover. Its activity was optimal at pH 4.5 and 50°, and it was strongly inhibited by glucono-1,5-lactone. The enzyme showed hydrolytic as well as transglycosylation activity towards (1 → 3)-β- and (1 → 4)-β-linked oligosaccharides with degree of polymerization of 2–4. The results suggest that the β-glucosidase is probably involved not only in hydrolysis but also in modification of oligosaccharides in cell walls of germinating rice seeds.

Solid Support Oligosaccharide Synthesis: Construction of β-Linked Oligosaccharides by Coupling of Glycal Derived Thioethyl Glycosyl Donors

A solution to the problem of a solid-phase synthesis of β-(1→4) linked glucosides is described. The method involves conversion of polymer bound glycal to polymer bound thioethyl glucosyl donor bearing an α-oriented pivaloxy group at C2. The latter directs couplings with solution-based glycal acceptors in the β-sense. The ready-reiteratability of the method was demonstrated.

Synthesis of Kdo-trisaccharide derivatives of chlamydial and enterobacterial LPS containing carboxyl-reduced or β-configurated Kdo-residues

Five allyl glycosides corresponding to the 3-deoxy-D- manno-2-octulosonic acid (Kdo) containing genus-specific LPS epitope of Chlamydia were synthesized. Compounds 5 and 22 contain one carboxyl-reduced Kdo moiety linked to O-4 of the proximal Kdo unit, whereas the analogues 28, 34 and 36 each contain one β-linked Kdo-residue within the trisaccharide sequence Kdo p-(2→8)-Kdo p-(2→4)-Kdo p. Elaboration of the carboxyl-reduced derivatives was achieved by BF3•Et2O-catalyzed coupling of Kdo-fluoride derivatives 1 or 14 with the 7,8- O-carbonyl-derivative 2. The β-linked oligosaccharides were obtained by Helferich-glycosidation of the respective Kdo-disaccharide bromide derivatives 26 and 31. The deprotected compounds - characterized by H and13C NMR spectroscopy - are suitable haptens for the immunochemical study of monoclonal antibodies directed against the Kdo-region of chlamydial and enterobacterial LPS.