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Microwave irradiation at different frequencies gave molecular selective effects, namely higher frequency microwave effects for waters while lower frequency effects for ions. We already reported that 2.45 GHz and 5.80 GHz microwave irradiation gave different results for a hydrolysis reaction by thermostable β-Glucosidase HT1. Here, we designed and made a reactor, employed 400 MHz microwave irradiation, and studied the effectiveness of 400 MHz microwave for HT1 reaction, then 400 MHz and 2.45 GHz had the ability to accelerate HT1 reaction. In consideration of the general mechanism of enzymatic glycoside hydrolysis, our results would be reasonable if ions are key reaction species because 400 MHz microwave activated ions selectively. In addition, the phenomenon that 400 MHz microwave would not affect water molecules by dielectric heating might contribute the enzyme stability. This report should support that microwave is not only a tool to heat reactions efficiently but also can bring unique effects for reactions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease. [ABSTRACT FROM AUTHOR]

Background: The Caldanaerobacter subterraneus species includes thermophilic fermentative bacteria able to grow on carbohydrates substrates with acetate and L-alanine as the main products. In this study, comprehensive analysis of three genomes of C. subterraneus subspecies was carried in order to identify genes encoding key metabolic enzymes and to document the genomic basis for the evolution of these organisms. Methods: Average nucleotide identity and in silico DNA relatedness were estimated for the studied C. subterraneus genomes. Genome synteny was evaluated using R2CAT software. Protein conservation was analyzed using mGenome Subtractor. Horizontal gene transfer was predicted through the GOHTAM pipeline (using tetranucleotide composition) and phylogenetic analyses (by maximum likelihood). Hydrolases were identified through the MEROPS and CAZy platforms. Results: The three genomes of C. subterraneus showed high similarity, although there are substantial differences in their gene composition and organization. Each subspecies possesses a gene cluster encoding a carbon monoxide dehydrogenase (CODH) and an energy converting hydrogenase (ECH). The CODH gene is associated with an operon that resembles the Escherichia coli hydrogenase hyc/hyf operons, a novel genetic context distinct from that found in archetypical hydrogenogenic carboxydotrophs. Apart from the CODH-associated hydrogenase, these bacteria also contain other hydrogenases, encoded by ech and hyd genes. An Mbx ferredoxin:NADP oxidoreductase homolog similar to that originally described in the archaeon Pyrococcus furiosus was uniquely encoded in the C. subterraneus subsp. yonseiensis genome. Compositional analysis demonstrated that some genes of the CODH-ECH and mbx operons present distinct sequence patterns in relation to the majority of the other genes of each genome. Phylogenetic reconstructions of the genes from these operons and those from the ech operon are incongruent to the species tree. Notably, the cooS gene of C. subterraneus subsp. pacificus and its homologs in C. subterraneus subsp. tengcongensis and C. subterraneus subsp. yonseiensis form distinct clades. The strains have diverse hydrolytic enzymes and they appear to be proteolytic and glycolytic. Divergent glycosidases from 14 families, among them amylases, chitinases, alpha-glucosidases, beta-glucosidases, and cellulases, were identified. Each of the three genomes also contains around 100 proteases from 50 subfamilies, as well about ten different esterases. Conclusions: Genomic information suggests that multiple horizontal gene transfers conferred the adaptation of C. subterraneus subspecies to extreme niches throughout the carbon monoxide utilization and hydrogen production. The variety of hydrolases found in their genomes indicate the versatility of the species in obtaining energy and carbon from diverse substrates, therefore these organisms constitute a remarkable resource of enzymes with biotechnological potential. [ABSTRACT FROM AUTHOR]

Background: α-L-Fucosidases are enzymes involved in metabolism of α-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which α-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed. Methods: Activity-based screening of a genomic library was used to isolate the gene encoding a novel α-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged α-L- fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation. Results: Using a genomic library of Paenibacillus thiaminolyticus,constructedin E.coli DH5α cells, nucleotide sequence of anew α-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine- tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of α-L-fucosidaseiso2weretestedusingdifferentacceptormolecules. Conclusions: In this study, new enzyme α-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known α-L-fucosidases was found, following study could be import- ant for different biochemical disciplines involving molecular modelling. [ABSTRACT FROM AUTHOR]

Glycosidases, including β- d-galactosidase and β- d-glucosidase, are involved in a range of metabolic disorders, such as cancer, viral or bacterial infections, and diabetes. Previously, we scanned the pharmacophoric space of these enzymes and had a self-consistent and predictive quantitative structure-activity relationship that was used to identify several β- d-galactosidase and β- d-glucosidase inhibitors via in silico search of structural databases. Guided by the preceding modeling efforts, synthesis of a series of tryptophan and thiosemicarbazide derivatives as β- d-galactosidase and β- d-glucosidase inhibitors that match the generated pharmacophores followed by in vitro bioassay was carried out. Synthesized compounds 3c (37 % inhibition at 100 µM) and 4d (49 % inhibition at 100 µM) exhibited the best inhibitory bioactivities against β- d-galactosidase and β- d-glucosidase, respectively. They can serve as a promising lead compounds for the development of potential glycosidase inhibitors. [ABSTRACT FROM AUTHOR]

The laccase-catalyzed oxidation of piceid, the 3-β- D-glucopyranosyl derivative of the stilbenic phytoalexin resveratrol, allowed isolation of the corresponding β-5 like trans-dehydrodimer in good yield (45%) avoiding chromatography. This compound was then submitted to the action of a small library of commercially available hydrolases. While the cellulase from Trichoderma viride was able to fully hydrolyze the diglycosylated dimer to give the corresponding β-5 like trans-dehydrodimer of resveratrol, the β-glucosidase from almonds allowed the isolation of a monoglycosylated intermediate in reasonable yield. The diastereoisomers and the enantiomers of the isolated dimeric products were separated using a semi-preparative chiral column. The basic antioxidant properties of these new dimers have been determined. [ABSTRACT FROM AUTHOR]

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermo-philus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-D-galactopyrano-syl-(1,6)-α-D-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases. [ABSTRACT FROM AUTHOR]

Aim: The aim of the study was to analyze the antioxidant, anti-amylase, anti-glucosidase and antiacetylcholinesterase (anti-AChE) properties of the leafy shoots of three Indian species of Swertia e.g. Swertia chirata and its substitutes Swertia bimaculata, and Swertia decussata, their xanthones and amarogentin. Methods: Antioxidant activity of the methanolic extracts of leafy shoots was measured in terms of DPPH, superoxide and nitric oxide radical scavenging activities as well as metal chelating properties. Enzyme inhibitory properties were measured using AChE, α-amylase and α-glucosidase respectively. Five xanthones bellidifolin (1), swerchirin (2), decussatin (3), mangiferin (4) and 1-hydroxy-3,5,8-trimethoxy xanthone (6) and one iridoid, amarogentin (5) were isolated from Swertia chirata. The activities of the isolated components were compared. Results: Swertia chirata exhibited best antioxidant and anti- AChE properties than the other two species. The plants also possessed α-glucosidase inhibitory properties but weak α-amylase inhibitory activity. Highest activities were observed in Swertia chirata. We report here, for the first time, the antioxidant, anti-AChE and anti-glycosidase activity of 1-hydroxy-3,5,8-trimethoxy xanthone. This xanthone had strongest DPPH radical scavenging activity and anti-AChE property. Conclusion: The results suggest the beneficial effects of the xanthones of Swertia chirata. But further study should be carried out to prove the efficacy in vivo. [ABSTRACT FROM AUTHOR]

A series of diastereomeric tetrahydroxylated azepanes featuring a carboxymethyl group at the pseudo-anomeric position have been synthesized from a common unsaturated intermediate. Syn - and anti -dihydroxylation reactions were achieved to yield the target compounds after efficient one-step deprotection of carbamate, ester and acetonide groups simultaneously. Screening of these polyhydroxylated azepanes toward a range of commercially available glycosidases was performed and one of the stereoisomers showed potent and selective inhibition toward β-galactosidase (IC 50 = 21 μM). [ABSTRACT FROM AUTHOR]

The enantiodivergent synthesis of polyhydroxylated pyrrolizidines has been achieved, starting from common intermediate 1 . The synthesis involved the stereoselective construction of the pyrrolizidine core unit by using intramolecular Michael cyclization reaction as the key reaction. The synthesized eight isomers were evaluated for various glycosidase inhibition effects. Compound 15 showed a moderate inhibitory activity against α- l -fucosidase and a high selectivity compared from the other glycosidases. [ABSTRACT FROM AUTHOR]

Expression of the genes encoding the enzymes involved in inulin, sucrose, and glucose metabolism in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus was studied. The exon-intron structure of the relevant genes was identified and the primers for quantitative PCR were optimized. Expression of the genes was found to depend on the carbon source. Glucose was shown to exhibit a repressive effect on inulinase synthesis by K. marxianus, while sucrose induced it, and in S. cerevisiae glucose and sucrose were inulinase inducer and repressor, respectively. [ABSTRACT FROM AUTHOR]

The availability of endogenous and dietary carbohydrates in the gastrointestinal tract influences the composition of the gut microbiota. Carbohydrate foraging requires the action of bacterially-encoded glycoside hydrolases, which release mono- and oligosaccharides taken up as carbon sources by multiple microbial taxa. In addition to providing nutrients to the microbiota, the cleavage of host glycans by bacterial glycoside hydrolases may alter the properties of surface glycoproteins involved in cell adhesion and activation processes in the gut lumen. To investigate the impact of bacterial glycoside hydrolase activities on the gut microbial composition and on host glycans during colon inflammation, we increased local glycoside hydrolase activity by supplementing mice with recombinant E. coli expressing specific sialidase, fucosidase and rhamnosidase enzymes during acute colitis induced by dextran sulfate sodium ingestion. Whereas increased fucosidase and rhamnosidase activity did not alter the course of colitis, increased sialidase activity exacerbated disease severity. The effect of increased sialidase activity on inflammation was not caused by changes in the microbial composition given that a similar shift in gut bacteria occurred in all groups of mice supplemented with recombinant E. coli. Increased sialidase activity in the colon of treated mice however significantly altered the distribution of sialic acid on mucosal glycans. Treatment of lamina propria dendritic cells with bacterial sialidase also strongly decreased the density of sialylated ligands to anti-inflammatory siglec lectins, indicating that the remodeling of surface sialylation caused by increased sialidase activity likely accounts for the observed exacerbation of acute colitis in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hasler, Tavares-Gomes, Gut, Swayambhu, Gysi, Hausmann, Arora and Hennet.)

Twenty-one commercial enzyme preparations used in winemaking were characterised for the α-L-rhamnosidase, α-L-arabinosidase, β-D-xylosidase, β-D-galactosidase, β-D-glucosidase, esterase, protease, cinnamoyl esterase and laccase activities. A new rapid fluorimetric method to assay esterase activity was developed. Enzyme preparations differed for the level of each enzyme activity assayed rather than for the type of enzymatic activity detected. High levels of protease, glycosidase, esterase and cinnamoyl esterase activity were found among enzyme preparations for different technological applications. A drastic reduction in the level of cinnamoyl esterase was observed in commercial grape juice, pH 3.6, total acidity 5.3 g L−1, sugar 170 g L−1. Protease activity was only weakly reduced, from 10 to 20%, in commercial grape juice. β-glucosidase activity levels are reduced in the presence of increasing concentration of glucose but are still present at the higher glucose concentration (100 g L−1). The extent of the reduction observed was dependent on the enzymatic preparations tested. [ABSTRACT FROM AUTHOR]

Bats are mammals of the order Chiroptera, which represents about 20% of all mammalian species worldwide. Bats have evolved various reproductive strategies, such as delayed sperm maturation, sperm storage, delayed fertilization, delayed implantation and delayed development. The strategies of delayed sperm maturation allow female bats, especially hibernating species, to reproduce successfully. Alpha-L-Fucosidase 1 gene (FUCA1) has been proved to be involved in the processes of sperm maturation and sperm-egg interactions in mammals. It is still not clear how the FUCA1 gene evolved in bats. Therefore, it is important to determine the evolutionary pattern of FUCA1 gene and positively selected sites which affect the reproductive activity of bats. In this work, we cloned the coding regions of FUCA1 from 7 species of bats and performed an evolutionary study of this gene. Although selection pressure varied among different lineages, FUCA1 gene underwent positive selection in the ancestral species leading to Old World fruit bats. Our results demonstrated that FUCA1 is basically conserved in hibernating bats but has evolved within the ancestral species of Old World fruit bats to adapt their survival environments. [ABSTRACT FROM AUTHOR]

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the −1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68–90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases. [ABSTRACT FROM PUBLISHER]

Glycosylation is one of the most important post-modification processes of small molecules and enables the parent molecule to have increased solubility, stability, and bioactivity. Enzyme-based glycosylation has achieved significant progress due to advances in protein engineering, DNA recombinant techniques, exploitation of biosynthetic gene clusters of natural products, and computer-based modeling programs. Our report summarizes glycosylation data that have been published within the past five years to provide an overall review of current progress. We also present the future trends and perspectives for glycosylation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Co-expression of human O-GlcNAc transferase and substrate proteins in Escherichia coli. [•] In vivo O-GlcNAc-modification of human CREB1 and ABL2 in E. coli. [•] β-N-acetylglucosaminidase inhibition increases O-GlcNAc levels. [•] E. coli NagZ deglycosylates O-GlcNAc-modified proteins in vivo. [•] NMR assay shows NagZ deglycosylates O-GlcNAc-modified peptide in vitro. [ABSTRACT FROM AUTHOR]

Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X-ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH-switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential., (© 2022 Wiley-VCH GmbH.)

Commonly found at the outermost ends of complex carbohydrates in extracellular medium or on outer cell membranes, sialic acids play important roles in a myriad of biological processes. Mammals synthesize sialic acid through a complex pathway, but Trypanosoma cruzi, the agent of Chagas’ disease, evolved to obtain sialic acid from its host through a trans-sialidase (TcTS) reaction. Studies of the parasite cell surface architecture and biochemistry indicate that a unique system comprising sialoglycoproteins and sialyl-binding proteins assists the parasite in several functions including parasite survival, infectivity and host-cell recognition. Additionally, TcTS activity is capable of extensively remodeling host cell glycomolecules, playing a role as virulence factor. This review presents the state of the art of parasite sialobiology, highlighting how the interplay between host and parasite sialic acid helps the pathogen to evade host defense mechanisms and ensure lifetime host parasitism.

Thanks to the stability, good availability, stereoselectivity and broad substrate specificity, oligosaccharide synthesis catalyzed by glycosidases represents an elegant way to complex carbohydrate structures. Two approaches to glycosidase catalysis are presented: i) the use of structurally modified substrates that carry various functional moieties in the molecule, and ii) the design of mutant glycosidases void of hydrolytic activity. Products of glycosidase-catalyzed synthesis are applicable in a range of are as such as immunology, therapy of Alzheimer's or Parkinson's diseases and the synthesis of neoglycoproteins.

This review covers the literature in the past 15 years on glycosidase inhibitors lacking a basic nitrogen (for example iminosugars/azasugars) with a focus on natural terpenoids, and mono- and polycyclic aromatic hydrocarbons. From quite diverse structures, insight into inhibitor structural features that may be applicable to optimisation of all glycosidase inhibitors including iminosugars are identified. [Display omitted] • Glycosidase inhibitors possessing terpenoid structures, and mono- and polycyclic aromatic hydrocarbon structures. • Benzimidazole, chalcone, cinnamic acid, coumarin, flavonoid, phthalimide, terpenoid, xanthone. • Rationalisation of potent glycosidase inhibition via a common structural motif. [ABSTRACT FROM AUTHOR]

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis of l-3-epi-isofagomine, its homo-, n-butyl derivatives and its bicyclic analogue as potent glycosidase inhibitor has been achieved from readily available d-glucose. Inhibition of some commercially available glycosidases was also carried out with the newly synthesized inhibitors which showed reasonably good inhibitions (9.4–198.2μM). One of them (compound 11) showed selective inhibition of β-galactosidase. [Copyright &y& Elsevier]

Human milk oligosaccharides (HMOS) are not digested in the proximal intestine. In distal intestine, HMOS collectively modify the microbiota, but the response of individual bacteria to individual components of the HMOS is not well defined. Here, each of 25 major isolates of the human intestinal microbiota was fed individual major fucosylated and sialylated HMOS in anaerobic culture. This allowed for an assessment of the influence of specific HMOS on the growth and metabolic products of individual microbiota bacteria. Most Bifidobacteria spp. and Bacteroides spp. grew, induced α-l-fucosidase activity, and produced abundant lactate or short-chain fatty acids (SCFAs) when fed 2′-fucosyllactose (2′-FL), 3-FL, and lactodifucotetraose (LDFT). Lactobacillus delbrueckii ATCC7830, Enterococcus faecalis ATCC19433, and Streptococcus thermophilus ATCC19258 exhibited slight growth, pH reduction, and lactate production when supplemented with 2′-FL or 3-FL, but not LDFT. Supplementation with 3′-sialyllactose (3′-SL) and 6′-SL promoted moderate growth of Bifidobacterium longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, Bacteroides vulgatus ATCC8482, and B. thetaiotaomicron ATCC29148; accordingly, these bacteria exhibited greater neuraminidase activity and produced copious lactate, SCFA, or both. Lactobacillus delbrueckii ATCC7830 also consumed 6′-SL. In contrast, Clostridium spp., L. rhamnosus ATCC53103, E. faecalis ATCC29200, Staphylococcus spp., Enterobacter spp., and Escherichia coli K12 did not consume milk oligosaccharides nor produce appreciable acidic fermentation products. Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated milk oligosaccharides most strongly stimulating key species of mutualist symbionts. This suggests strategies for treating dysbiosis of the microbiota and associated inflammatory disorders. [ABSTRACT FROM PUBLISHER]

Abstract: Background: The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases. Methods: The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated. Results: Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase. Conclusions: The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases. General significance: The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases. [Copyright &y& Elsevier]

1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy ( STD NMR), a comparison is made between wild-type xylanase XT6 and its acid/base catalytic mutant E159Q - a non-active, single-heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide-xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275-11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose-xylanase complexes, a ' SASSY' plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non-covalent interactions with H2-5 of xylobiose were observed in the wild-type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β-xylobiose anomer. The 1H solution NMR spectrum of a xylotriose-E159Q complex displays non-uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face-to-face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate-observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate-binding adjustments upon a charge change in the E159Q construct. [ABSTRACT FROM AUTHOR]

Abstract: Fucosidases, enzymes that cleave fucose from the non-reducing end of a glycan, represent promising medicinal targets reflecting their roles in cancer metastasis, inflammation, host-parasite interactions and the lysosomal storage disorder fucosidosis. The X-ray crystal structures of Bacteroides thetaiotaomicron GH29 α-l-fucosidase (BtFuc2970) in a new crystal form (at a resolution of 1.59Å) and liganded with a 5-membered iminocyclitol inhibitor (1.73Å) are reported herein. The 5-membered iminocyclitol binds in a 3E conformation, mimicking the proposed 3H4 half chair transition-state of the enzyme catalysed reaction, and its K i for BtFuc2970 was determined as 2μM. Structural analysis of fucosidase inhibition through 5-membered iminocyclitols will aid in the rational design of more potent fucosidase inhibitors for treatment of a range of medical conditions. [Copyright &y& Elsevier]

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20( S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml ginsenoside Rb2 was transformed into 0.47 mg ml ginsenoside 20( S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20( S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20( S)-Rg3 using the recombinant glycosidase. [ABSTRACT FROM AUTHOR]

A significant number of terpenoid compounds are glycosides with the sugars linked to the active groups. Sometimes, the glycosidic residue is crucial for their activity, but in other cases glycosylation only improves pharmacokinetic parameters. Enzymatic glycosylation of terpenoids is a useful tool due to the high selectivity and the mildness of the reaction conditions, in comparison with chemical methods. Several types of biocatalysts have been used in the enzymatic glycosylation of terpenoids. These include the use of glycosyltransferases, trans-glycosidases, and whole-cell biotransformation systems capable of regenerating the cofactor, such as fungi, bacteria, plant-cell cultures, etc. Many biosynthesized terpenoid glycosides display medicinal and pharmacological properties and can be used as pro-drug substances. These terpenoid glycosides have also been employed as food additives (e.g. low-caloric sweetener compounds) and cosmetics, and even have applications as controlled-release fragrances. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of two per-O-methylated cyclodextrins with one or two α-hydroxypropionitrile groups attached to the secondary rim is described. The two new cyanohydrins are found to catalyse the hydrolysis of 4-nitrophenyl β-D-glucopyranoside in a buffer solution at pH 8.0 with a rate acceleration of up to 1770. [Copyright &y& Elsevier]