D-glucose Residues Research Articles

Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active Against Multidrug Resistant Tuberculosis.

Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, particularly their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinomycetota extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 μM. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster (BGC). A cytochrome P450 encoded by this gene cluster catalyzes the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyze the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. One of these encodes a glycosyl transferase that was shown to catalyze attachment of a D-glucose residue to nosiheptide in vitro. The discovery of the persiathiacins and their BGC thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

Molecular mechanism for the substrate specificity of Arthrobacter globiformis M6 α-glucosidase CmmB, belonging to glycoside hydrolase family 13 subfamily 30

α-Glucosidase hydrolyzes α-d-glucosides to produce α-d-glucose. Glycoside hydrolase family 13 (GH13) contains α-glucosidases together with various amylolytic enzymes. GH13 α-glucosidases fall into several distinct subfamilies, and subfamily 30 (GH13_30) includes numerous Actinomycetes α-glucosidases. GH13_30 α-glucosidase CmmB from Arthrobacter globiformis, specific to maltooligosaccharides, is involved in the intracellular metabolism of cyclobis-(1 → 6)-α-maltosyl. Herein, the function and structure of CmmB were investigated to advance understanding of the structure–function relationship. CmmB showed the highest kcat/Km for maltose of maltooligosaccharides, and kcat/Km drastically decreased with increasing substrate chain-length. The crystal structures of CmmB in complex with a pseudodisaccharide (acarviosin) and pseudotetrasaccharide (acarbose) were determined at resolutions of 1.60 and 1.70 Å, respectively. The overall structure of CmmB was typical of a GH13 α-glucosidase. Most of the structure of the β→α loop 7 of the catalytic (β/α)8-barrel domain was not determined in the acarbose complex, but the C-terminal side of this loop was modeled in the acarviosin complex. For binding to acarviosin, this loop took on a closed conformation, and I360 and R364 on this loop formed subsite +1 together with H224 and W280. Alanine substitution of these residues indicated that R364 was essential for the catalysis through a hydrogen bond with the O6 of the d-glucose residue in subsite +1. The β→α loop 7 is flexible upon binding to substrates, but I360 and R364 cannot participate in binding to maltooligosaccharides longer than maltose. The loss of interactions with these residues was concluded to result in the low preference for maltotriose and longer maltooligosaccharides.

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on β→α Loop 5.

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

Gellan gum biopolymer- A review

Gellan gum is an anionic polysaccharide produced by the bacterium Sphingomonas paucimobilis. Kaneko and Kang discovered the biopolymer in the laboratory of the Kelco Division of Merck and Co., California, USA. It is composed of tetrasaccharide repeating units of two residues of D-glucose, one of D-glucuronic and one of L-rhamnose. The functional properties of gellan gum make it one of the industrially useful exopolysaccharides. Sphingomonas paucimobilis ATCC 31461 is the bacterium used for the industrial production of gellan gum. The gellan gum has potential applications in food, pharmaceutical, biomedical and other industries. The gellan gum finds uses in the bioremediation field. Bioaugmentation of contaminated aquifers, biodegradation of hydrocarbons and the removal of dyes from wastewater are a few of its applications. This review provides an overview of the characteristics, production of gellan gum and recent bioremediation applications.

Исследование сорбционной способности растворимых форм дрожжевого бета-глюкана

Beta-glucans are polysaccharides that consist of D-glucose residues linked together in the main and side chains by glycosidic bonds. They are obtained from plant and microbial raw materials. Beta-glucans obtained from various sources differ in molecular weight, backbone length, branching of additional chains and their number. Beta-(1,3,1,6)-glucan from the yeast Saccharomyces cerevisiae has the highest physiological activity. The soluble fraction of beta-glucan has a higher physiological activity than its insoluble fraction. As a rule, soluble beta-glucans appear to be more potent immunostimulants than insoluble ones. In this regard, obtaining soluble forms of beta-glucan from yeast is relevant. The purpose of this work was to study the effect of the content of the soluble fraction in yeast beta-glucan on the ability to adsorb heavy metal ions, mycotoxins and cholesterol. Evaluation of the effectiveness of ultrasonic treatment for obtaining soluble forms of yeast beta-glucan was carried out. It was found that the maximum content of the soluble fraction, equal to 95%, at a frequency of ultrasonic treatment of 85 kHz for a laboratory sample of beta-glucan, is achieved with a treatment time of 20 min, and for a commercial one – in 30 min. The sorption properties of soluble forms of yeast beta-glucan with different content of the soluble fraction in relation to cholesterol, aflatoxin and bivalent copper cations were studied. The sorption capacity of samples of laboratory and commercial preparations of beta-glucan was determined for the above compounds. It was found that an increase in the content of the soluble fraction to more than 50% does not lead to a noticeable increase in the sorption capacity. It was shown that purified samples of beta-glucan have higher sorption characteristics.

Isolation and characterization of dextran produced by Lactobacillus sakei L3 from Hubei sausage

An exopolysaccharide (EPS)-producing bacterial strain L3 was isolated from Hubei sausage and identified as Lactobacillus sakei via morphological, physiological, biochemical and 16S rDNA analysis. FT-IR spectroscopy and NMR revealed that L3 EPS was a dextran containing d-glucose residues with α-1,6 glycosidic linkage. Rheological studies showed that it had high viscosity at high concentration, low temperature, and acidic pH (pH 3.0). Scanning electron microscopy of the L3 dextran demonstrated a porous and branched morphology, and atomic force microscopy showed lumps of varying height on the rough surface of the L3 EPS polymer. The EPS was thermally stable up to 272°C and could coagulate sucrose-supplemented milk. Together, these results suggested that L3 EPS might have potential applications in food processing and other areas.

Structural insights into antigen recognition of an anti-\u03b2-(1,6)-\u03b2-(1,3)-D-glucan antibody

Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related β-(1,6)-branched β-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched β-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27H, His35H, Trp47H, Trp100H, Asp105H; Asp49L, Lys52L, Trp90L) that contribute to the recognition of glucose units from all three chains of the SCH triple helix. The importance of these amino acids was confirmed by mutagenesis and ELISA-based analysis. Our work provides an explanation for the specific recognition of triple-helical β-(1,6)-branched β-(1,3)-D-glucans by JoJ48C11 and provides another structure example for anti-carbohydrate antibodies.

Cooperative Order–Disorder Transition of Carboxylated Schizophyllan in Water–Dimethylsulfoxide Mixtures

Carboxylated schizophyllan ("sclerox") is a chemically modified polysaccharide obtained by partial periodate oxidation and subsequent chlorite oxidation of schizophyllan, a water-soluble neutral polysaccharide having a β-1,3-linked glucan backbone and a β-1,6-linked d-glucose residue side chain at every third residue of the main chain. The triple helix of schizophyllan in water has a cooperative order-disorder transition associated with the side chains. The transition is strongly affected by the presence (mole fraction) of dimethylsulfoxide (DMSO). In the present study, the solvent effects on the order-disorder transition of sclerox with different degrees of carboxylation (DS) in water-DMSO mixtures were investigated with differential scanning calorimetry and optical rotation. The transition temperature ( Tr) and transition enthalpy (Δ Hr) strongly depended on the mole fraction of DMSO ( xD). Data were further analyzed with the statistical theory for the linear cooperative transition, taking into account the solvent effect, where DMSO molecules are selectively associated with the unmodified side chains. The modified side chain does not contribute to the transition; hence, Δ Hr decreases with increasing DS. The dependence of Tr on the DMSO content becomes weaker than that for unmodified schizophyllan. The theoretical analyses indicated that the number of sites binding with the DMSO molecule and the successive ordered sequence of the ordered unit of the triple helix are changed by carboxylation.

Chemoenzymatic synthesis of sucuronic acid using d-glucurono-6,3-lactone and sucrose as raw materials, and properties of the product.

Using d-glucurono-6,3-lactone (GlcL) and sucrose (Suc) as raw materials, we synthesized sucuronic acid (SucA), in which the d-glucose (Glc) residue of Suc was replaced with d-glucuronic acid, by a three-step chemoenzymatic method. In the 1st chemical step, methyl d-glucuronate (GlcAM) was synthesized by treating GlcL with a strong base anion exchange resin, Amberlite IRA402BL OH AG, in anhydrous methanol. In the 2nd step, which included an enzyme reaction, methyl sucuronate (SucAM) was synthesized from GlcAM and fructose by exploiting the transfructosylation activity of the Microbacterium saccharophilum K-1 β-fructofuranosidase, a reaction that is suppressed in the presence of high-concentration Glc. In this reaction, the addition of a Suc-non-assimilating yeast, Saccharomyces bisporus NBRC1131, to the reaction mixture increased the amount of SucAM generated, because Glc was removed from the mixture by this yeast. In the 3rd chemical step for producing sodium sucuronate (SucA·Na), SucAM was treated with Amberlite IRA402BL OH AG in water to hydrolyze SucAM's ester bond, and product was then treated with NaOH. The molar yield of SucA·Na from GlcL was 34.2%. SucA was stable at 37 °C in buffer solutions at pH 3, 5, 7, or 9. However, at temperatures exceeding 75 °C, the glycosidic bond of this disaccharide was hydrolyzed not only in acidic buffers (pH 3 and 5) but also in alkaline buffer (pH 9). SucA was not a suitable substrate for the β-fructofuranosidases of M. saccharopilum K-1 and Saccharomyces cerevisiae.

Use of Lentinan To Control Sharp Eyespot of Wheat, and the Mechanism Involved.

Lentinan (LNT), a complex polysaccharide with a β-(1→3)-linked backbone of d-glucose residues, has been reported to inhibit plant diseases. Our objective was to explore the efficacy and action mechanism of LNT used as a seed dressing to control sharp eyespot of wheat. Seed dressing promoted wheat growth. At control germination rates of 50%, 8 g of LNT/100 kg of seeds of the Jimai 22, Shannong 23, and Luyuan 502 cultivars significantly increased seed germination to 54%, 52%, and 51%, respectively. Seven days after emergence, the heights and root activity of wheat treated with LNT were significantly greater than those of controls. These effects were dose-dependent. At this time, the plant heights of Jimai 22, Shannong 23, and Luyuan 502 cultivars were 9.52, 8.52, and 10.52 cm, respectively, significantly higher than that of the controls. LNT prevented the development of wheat sharp eyespot. In the highly susceptible Jimai 22 cultivar, sharp eyespot development was reduced by 33.7%, 31.9%, and 30.4% at 7, 14, and 21 days after germination. LNT somewhat increased phenylalanine ammonia-lyase, peroxidase, and superoxide dismutase activity; reduced the malondialdehyde content; increased chlorophyll a and b levels; and enhanced the root vigor of wheat. These effects peaked 7 days after germination. LNT increased transcription of the genes encoding alternative oxidase (AOX) and β-1,3-glucanase (GLU), the salicylic acid signaling pathway-related gene NbPR1a, and the sharp eyespot resistance-related gene RS33. A significant dose-effect relationship was evident in terms of AOX transcription; we thus speculate that AOX may be the target gene.

Characterization of branched limit dextrin and impact on corn starch pasting properties

Branched limit dextrins (BLDs) are composed of linear chains of alpha-(1,4)- d-glucose residues connected through alpha-(1,6)-linkages. In this work, BLDs were prepared by hydrolysis of starch by alpha- and beta-amylases and subsequent ethanol precipitation into fractions of different molecular weights and structures. The characterization of BLDs was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 1H nuclear magnetic resonance spectroscopy. The average molecular weights of BLDs from both corn and waxy corn starches decreased upon an increase in ethanol concentration from 70% to 90%, that is, from 1918.30 to 1274.23 and from 1938.11 to 1356.35, while the ratio of alpha-(1,4) to alpha-(1,6) glycosidic linkages increased from both corn (from 5.27 to 8.46) and waxy corn starches (from 3.80 to 4.62). The effect of BLDs with different degrees of polymerization on the pasting properties of corn starch was investigated by rapid visco-analysis (RVA). Confocal laser scanning microscopy (CLSM) observation of the BLD/cornstarch mixtures was also carried out to explore the possible interaction between corn starch and BLDs. RVA showed that BLDs significantly changed the viscosity parameters of the BLD/cornstarch mixtures. The results of CLSM observation of samples further indicate that the cornstarch and BLDs interacted in the cornstarch–BLD system because of the low molecular weight and mobility of BLDs.

Characteristic of Polysaccharide Complexes from Centaurea scabiosa L. and Centaurea pseudomaculosa Dobrocz.

We characterized polysaccharide complexes from Centaurea scabiosa L. and Centaurea pseudomaculosа Dobrocz. We proposed the technique of sequential selection of water-soluble polysaccharides and pectin substances from the aerial parts of studied objects. We have discovered that the content of water-soluble polysaccharides in the aerial parts of C. scabiosa was 2.8 times higher (2.7 ± 0.3%, n = 3) than in C. pseudomaculosа (0.97 ± 0.50%, n = 3). The content of pectin substances in the aerial parts of C. scabiosa was 2 times higher (7.6 ± 0.4%, n = 3) than in C. pseudomaculosа (3.9 ± 0.3%, n = 3). The residues of D-galacturonic acid, L-rhamnose, D-xylose, D-mannose, D-glucose, and D-galactose are the monomeric units of polysaccharide complexes from C. scabiosa and C. pseudomaculosa. Using ion-exchange chromatography, three polysaccharide fractions (molecular weights 667, 722, and 1027 kDa), whose monomer units are D-galacturonic acid, L-rhamnose, D-galactose, D-xylose, and D-glucose were isolated from the water-soluble polysaccharides of C. scabiosa.

Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus

BackgroundLactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization.ResultsUsing superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used.ConclusionThese results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

Characterization of a dextran produced by Leuconostoc pseudomesenteroides XG5 from homemade wine

A water-souble exopolysaccharide (EPS) produced by Leuconostoc pseudomesenteroides XG5 from homemade wine was investigated. The EPS yield of 35.5g/L was achieved at 30°C for 48h in De Man-Rogosa-Sharpe (MRS) medium containing 12.5% sucrose. The EPS was a dextran composed exclusively of glucose and the molecular weight was 2.6×106Da. Fourier transform infrared spectra and nuclear magnetic resonance spectra revealed that the EPS was a dextran containing D-glucose residues in a linear chain with consecutive α-(1→6) linkages. Scanning electron microscopy of the EPS appeared a highly branched and porous structure. Rheological studies showed that the EPS had higher viscosity in 0.1M KCl solution, at lower temperature, or at acidic pH. Thermal gravimetric analysis and differential scanning calorimetric indicated that the EPS had excellent thermal stability with a degradation temperature of 313.80°C and melting point at 274.14°C. Water solubility index and water holding capacity of XG5 dextran were 90.2% and 412% respectively. The results suggest that L. pseudomesenteroides XG5 might be widely used in the production of linear dextran which has potential to serve as natural agent applied in food and other fields.

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase.

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two D-glucose units, such as D-maltose or D-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH2CHCOOH loss via cleavage of the Cα-Cβ bond in Trp induced by hydrogen atom transfer from the NH3+ group of a protonated Trp was observed in a noncovalent heterochiral H+(L-Trp)(D-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H+(D-Trp)(D-maltose). For D-cellobiose, both NH2CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links D-glucose residues to the indole moiety of Trp via a C-C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.

A Broad-Host-Range Tailocin from Burkholderia cenocepacia.

ABSTRACTThe Burkholderia cepacia complex (Bcc) consists of 20 closely related Gram-negative bacterial species that are significant pathogens for persons with cystic fibrosis (CF). Some Bcc strains are highly transmissible and resistant to multiple antibiotics, making infection difficult to treat. A tailocin (phage tail-like bacteriocin), designated BceTMilo, with a broad host range against members of the Bcc, was identified in B. cenocepacia strain BC0425. Sixty-eight percent of Bcc representing 10 species and 90% of non-Bcc Burkholderia strains tested were sensitive to BceTMilo. BceTMilo also showed killing activity against Pseudomonas aeruginosa PAO1 and derivatives. Liquid chromatography-mass spectrometry analysis of the major BceTMilo proteins was used to identify a 23-kb tailocin locus in a draft BC0425 genome. The BceTMilo locus was syntenic and highly similar to a 24.6-kb region on chromosome 1 of B. cenocepacia J2315 (BCAL0081 to BCAL0107). A close relationship and synteny were observed between BceTMilo and Burkholderia phage KL3 and, by extension, with paradigm temperate myophage P2. Deletion mutants in the gene cluster encoding enzymes for biosynthesis of lipopolysaccharide (LPS) in the indicator strain B. cenocepacia K56-2 conferred resistance to BceTMilo. Analysis of the defined mutants in LPS biosynthetic genes indicated that an α-d-glucose residue in the core oligosaccharide is the receptor for BceTMilo.IMPORTANCE BceTMilo, presented in this study, is a broad-host-range tailocin active against Burkholderia spp. As such, BceTMilo and related or modified tailocins have potential as bactericidal therapeutic agents against plant- and human-pathogenic Burkholderia.

In vitro anticancer activity of the laminarans from Far Eastern brown seaweeds and their sulfated derivatives

Brown seaweeds have drawn worldwide attention due to their involvement in many industrial applications. They produce neutral low molecular weight polysaccharides (laminarans) with beneficial biological activities. However, the anticancer activity and molecular mechanism of the laminarans and their sulfated derivatives have not been investigated in detail. Herein, the laminarans from brown seaweeds Saccharina cichorioides, Saccharina japonica, and Fucus evanescens were isolated. The laminarans from S. cichorioides and S. japonica were confirmed to contain a main chain of β-(1→3)-D-glucopyranose with single branches at C6. The laminaran from F. evanescens consisted of not only β-(1→3)-linked D- glucopyranose but also includes single β-(1→6)-linked D-glucose residues. The branches at C6 are presented as glucose or as gentiobiose. The sulfated laminarans with different degrees of sulfation were obtained by the chlorosulfonic acid-pyridine assay. In modified polysaccharides, the positions of sulfates are directly predetermined by the structure of native laminarans. The laminarans and their sulfated derivatives inhibited proliferation, colony formation, and migration of human colorectal adenocarcinoma, melanoma, and breast adenocarcinoma cells in different manners. The sulfated laminaran from F. evanescens possessed the highest anticancer activity in vitro and effectively prevented migration of breast adenocarcinoma cells by inhibiting of the Matrix Metalloproteinases 2 and 9 activity.

Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases.

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.

Toward a Molecular Lego Approach for the Diversity-Oriented Synthesis of Cyclodextrin Analogues Designed as Scaffolds for Multivalent Systems.

A modular strategy has been developed to access a diversity of cyclic and acyclic oligosaccharide analogues designed as prefunctionalized scaffolds for the synthesis of multivalent ligands. This convergent approach is based on bifunctional sugar building blocks with two temporarily masked functionalities that can be orthogonally activated to perform Cu(I)-catalyzed azide-alkyne cycloaddition reactions (CuAAC). The reducing end is activated as a glycosyl azide and masked as a 1,6-anhydro sugar, while the nonreducing end is activated as a free alkyne and masked as a triethylsilyl-alkyne. Following a cyclooligomerization approach, the first examples of close analogues of cyclodextrins composed of d-glucose residues and triazole units bound together through α-(1,4) linkages were obtained. The cycloglucopyranoside analogue containing four sugar units was used as a template to prepare multivalent systems displaying a protected d-mannose derivative or an iminosugar by way of CuAAC. On the other hand, the modular approach led to acyclic alkyne-functionalized scaffolds of a controlled size that were used to synthesize multivalent iminosugars.

Structure of Ulvan Isolated from the Edible Green Seaweed, Ulva pertusa

Ulvan, rhamnan sulfate, was extracted from the edible green seaweed, Ana-aosa (Ulva pertusa), which is grown on the coast of the Okinawa Islands. The yield of ulvan was 8.5% (W/W), and the total carbohydrates, uronic acid and sulfuric acid and ash contents were 67.3%, 23.8%, 19.7% and 22.6%, respectively. L-Rhamnose, D-xylose and D-glucose residues were identified by liquid chromatography, and their molar ratio was 4.0:0.1:0.3. D-Glucuronic and L-idulonic acid residues were also identified in molar ratio of 1.0:0.2. The NMR (13C and 1H) and methylation analysis revealed terminal β-D-glucruonic acid, terminal α-L-idulonic acid, 1,3-linked α-L-rhamnose, 1,4-linked α-L-rhamnose, 1,2,4-linked α-L-rhamnose, 1,3,4-linked α-L-rhamnose, 1,2,3,4-linked α-L-rhamnose and 1,3,4-linked β-D-xylose. The sulfate groups were attached at the C-2 and C-3 positions of the 1,4-linked α-L-rhamnose as well as C-3 of the 1,4-linked β-D-xylose residues. The chemical structure of the ulvan from Ulva pertusa was determined.