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When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Within the kingdom of fungi, the division Basidiomycota represents more than 30,000 species, some with huge genomes indicating great metabolic potential. The fruiting bodies of many basidiomycetes are appreciated as food (“mushrooms”). Solid-state and submerged cultivation processes have been established for many species. Specifically, xylophilic fungi secrete numerous enzymes but also form smaller metabolites along unique pathways; both groups of compounds may be of interest to the food processing industry. To stimulate further research and not aim at comprehensiveness in the broad field, this review describes some recent progress in fermentation processes and the knowledge of fungal genetics. Processes with potential for food applications based on lipases, esterases, glycosidases, peptidases and oxidoreductases are presented. The formation and degradation of colourants, the degradation of harmful food components, the formation of food ingredients and particularly of volatile and non-volatile flavours serve as examples. In summary, edible basidiomycetes are foods—and catalysts—for food applications and rich donors of genes to construct heterologous cell factories for fermentation processes. Options arise to support the worldwide trend toward greener, more eco-friendly and sustainable processes.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Bacteria biofilms consisting of surface-attached bacterial communities embedded in an extracellular matrix serve as a defense mechanism for many medically important bacterial species. Exopolysaccharides of partially de-N-acetylated poly-β-D-(1->6)-N-acetyl-glucosamine (dPNAG) are key structural components in the biofilm of many human pathogens. Dispersin B (DspB), a family 20 glycoside hydrolase produced by the Aggregatibacter actinomycetemcomitans, catalyzes the hydrolysis of dPNAG to disrupt biofilm formation leading to its use as an aniti-biofilm agent. Yet little is known of substrate recognition by DspB.Here, we describe the synthesis of two series of PNAG trisaccharide analogs with defined N-acetylation pattern (2.1 – 2.5) or containing glucose moiety (2.32 and 2.33) prepared through an iterative one-pot glycosylation approach and used to profile the activity and substrate preference of DspB (Chapter 2). These studies suggest that DspB hydrolyzes dPNAG polysaccharides via both exo- or endoglycosidase mechanisms and has a substrate preference for cationic substrates at the +2 position of the binding site. Understanding the activity and specificity of DspB provides a valuable guide to develop biocatalyst with improved biofilm dispersal activity. Next, colorimetric (Chapter 3) and fluorogenic (Chapters 4 & 5) PNAG analogs were developed as substrates for high-throughput PNAG glycosidase assay development. PNAG disaccharide probes (3.1 and 5.1) demonstrate exclusive specificity for enzymes capable of hydrolyzing PNAG and monosaccharide analog AMC-GlcNAc (4.1) acts as a general hexosaminidases enzyme substrate. We showed that all the analogs can detect DspB activity in crude E. coli cell lysates, and thus could be applied for functional metagenomic screening to discover novel PNAG glycosidase enzyms. Finally, a series of PNAG triazinyl glycosides (6.1, 6.2 and 6.3) were designed, synthesized and evaluated as affinity labeling reagents for PNAG binding proteins, usi

This study aimed to recover metagenome-assembled genomes (MAGs) from human fecal samples to characterize the glycosidase profiles of Bifidobacterium species exposed to different prebi-otic oligosaccharides (galacto-oligosaccharides, fructo-oligosaccharides and human milk oligosac-charides, HMOs) as well as high-fiber diets. A total of 1806 MAGs were recovered from 487 infant and adult metagenomes. Unsupervised and supervised classification of glycosidases codified in MAGs using machine-learning algorithms allowed establishing characteristic hydrolytic profiles for B. adolescentis, B. bifidum, B. breve, B. longum and B. pseudocatenulatum, yielding classification rates above 90%. Glycosidase families GH5 44, GH32, and GH110 were characteristic of B. bifidum. The presence or absence of GH1, GH2, GH5 and GH20 was characteristic of B. adolescentis, B. breve and B. pseudocatenulatum, while families GH1 and GH30 were relevant in MAGs from B. longum. These characteristic profiles allowed discriminating bifidobacteria regardless of prebiotic exposure. Correlation analysis of glycosidase activities suggests strong associations between glycosidase families comprising HMOs-degrading enzymes, which are often found in MAGs from the same species. Mathematical models here proposed may contribute to a better understanding of the carbohydrate metabolism of some common bifidobacteria species and could be extrapolated to other microorganisms of interest in future studies.

Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry.

[EN] Microalgae have features that make them ideal to produce biofuels. For instance, their starch can be used for bioethanol production. However, its degradation by glycosidases as soluble enzymes is expensive. To reduce its costs, biocatalysts can be easily recovered if they are immobilized as magnetic Cross-Linked Enzyme Aggregates (mCLEAs). This work helped characterise degradation of microalgal extracts by two glycosidases (alpha-amylase and amyloglucosidase) as soluble enzymes and as mCLEAs. Specifically, the effect of a thermal pretreatment of the microalgal extract, activation of alpha-amylase by calcium ions, and the optimum pH of activity for both types of mCLEAs was studied.

peer-reviewed, no abstract available, PUBLISHED, peer-reviewed

[EN] Microalgae have features that make them ideal to produce biofuels. For instance, their starch can be used for bioethanol production. However, its degradation by glycosidases as soluble enzymes is expensive. To reduce its costs, biocatalysts can be easily recovered if they are immobilized as magnetic Cross-Linked Enzyme Aggregates (mCLEAs). This work helped characterise degradation of microalgal extracts by two glycosidases (alpha-amylase and amyloglucosidase) as soluble enzymes and as mCLEAs. Specifically, the effect of a thermal pretreatment of the microalgal extract, activation of alpha-amylase by calcium ions, and the optimum pH of activity for both types of mCLEAs was studied.

[EN] Microalgae have features that make them ideal to produce biofuels. For instance, their starch can be used for bioethanol production. However, its degradation by glycosidases as soluble enzymes is expensive. To reduce its costs, biocatalysts can be easily recovered if they are immobilized as magnetic Cross-Linked Enzyme Aggregates (mCLEAs). This work helped characterise degradation of microalgal extracts by two glycosidases (alpha-amylase and amyloglucosidase) as soluble enzymes and as mCLEAs. Specifically, the effect of a thermal pretreatment of the microalgal extract, activation of alpha-amylase by calcium ions, and the optimum pH of activity for both types of mCLEAs was studied.

Use of the 3,5-dinitrosalicylic acid reagent allows the simple and rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in the characterization of enzyme reactions. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance and sugar concentration., Funding details: Knut och Alice Wallenbergs Stiftelse; Funding details: KTH, Kungliga Tekniska Högskolan; Funding details: Svenska Forskningsrådet Formas; Funding text: The author thanks the support of the Knut and Alice Wallenberg Foundation through the Wallenberg Wood Science Centre, Sweden, as well as the KTH Carbohydrate Materials Consortium (CarboMat) supported by the Swedish Research Council FORMAS.QC 20171108

A notable need exists for noninvasive tools to increase our mechanistic understanding of disease progression at a cellular and molecular level. Studying the functions of proteins in their innate in vivo tissue environment can provide useful information about pathology enabling appropriate treatment and early diagnosis. Chemical exchange saturation transfer MRI contrast provides real-time functional characterization of the biological landscape and can be used to detect multiple enzyme biomarker activities. A dual-enzyme catalyCEST contrast agent was developed as a proof-of-concept to demonstrate the potential of using a salicylic acid scaffold and control the CEST signal through enzyme activation. In addition, a straightforward route was designed to synthesize a diamagnetic catalyCEST MRI agent that is a substrate for β-galactosidase and β-glucuronidase enzymes. The synthesized agents generated two peaks in the CEST spectrum, at 4.25 ppm corresponding to a carbamate moiety and at 9.25 ppm corresponding to the salicylic acid moiety. Chemical exchange rates of liable protons were determined from a QUESP Hanes-Woolf plot. In the presence of the corresponding enzymes, the catalyCEST agent was activated via saccharide hydrolysis followed by a spontaneous disassembly to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST signal at 4.25 ppm. The CEST signal at 9.25 ppm was unaffected by the enzyme catalysis, and therefore used as an internal control signal. Michaelis-Menten enzyme kinetics studies were performed with CEST MRI to verify that catalyCEST MRI could truly detect enzyme activity. The Michaelis-Menten kinetics constants from MRI studies were compared to the kinetics constants measured with UVvis results from the same contrast agent, demonstrating the quantitative potential of catalyCEST MRI with both contrast agents. These findings demonstrate that the newly synthesized modular agents hav

[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 f

With recent pressure to reduce the environmental impact of leather production, research has been focused on the development of an alternative depilation method, as the conventional method for depilation contributes up to 60% of the total pollution produced. Contaminated salted ovine pelts stored at LASRA were easily depilated when drum washed, and the resultant leather was of good quality. The pelts were visibly contaminated with microorganisms, and it was thought that these may be secreting enzymes that loosened the wool fibre without damaging key structural skin components. Identification of the enzyme or enzymes was thus of interest. The microorganism/s responsible for the secretion of the depilation enzyme/s were isolated and identified through sequencing the 16S/18S ribosomal RNA genes. Depilation, using the crude secretome solutions, was then assessed using fresh ovine skin as well as SACPIC, a micro scale staining method used to assess skin structure. Unfortunately, none of the secretomes from either a single or a combination of the microorganisms isolated, had depilation activity. The secretome of W.ichthyophaga, a xerophilic filamentous fungus, which was consistently isolated from the contaminated pelts, was chosen to be characterised using proteomic methods. 1D SDS-PAGE gel/CHIP separation of the proteins in the secretome showed it contained mainly glycosidases, with no lipases, esterases, or proteases identified. Some of the proteins identified had suggested roles in resistance to osmotic pressure, while the remaining proteins were intracellular. Overall, 21 proteins were identified. A purification procedure involving AEX and SEC was successfully developed for the isolation of one of the glycosidases from the secretome. The resultant purified fractions formed a doublet band when analysed by SDS-PAGE. The reason for this remains unknown, but was shown not to be due to an impurity or heterodimerisation. The purified glycosidase was identifed as belonging to

The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining -glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido--cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI= 1 M).

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a S-1(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones., QC 20100525. QC 20211018.

The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of α-amylase, glucoamylase and α-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative α-glucosidase (AgdB) and an α-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative α-glucan modifying processes are discussed. © 2008 The Author(s).

The hydrolysis of toxic 7-hydroxycoumarin glucosides and other aryl and alkyl glucosides, catalyzed by modified a- and ß-cyclodextrin dicyanohydrins, was investigated using different UV, redox, or HPAEC detection assays. The catalyzed reactions all followed Michaelis-Menten kinetics, and an impressive rate increase of up to 7569 (kcat/kuncat) was found for the hydroxycoumarin glucoside substrate 4-MUGP. Good and moderate degrees of catalysis (kcat/kuncat) of up to 1259 were found for the natural glucosides phloridzin and skimmin. By using a newly developed catechol detection UV-assay, a weak degree of catalysis was also found for the toxic hydroxycoumarin esculin. A novel synthesized diaminomethyl ß-cyclodextrin showed a weak catalysis of p-nitrophenyl ß-D-glucopyranoside hydrolysis.

The hydrolysis of toxic 7-hydroxycoumarin glucosides and other aryl and alkyl glucosides, catalyzed by modified a- and ß-cyclodextrin dicyanohydrins, was investigated using different UV, redox, or HPAEC detection assays. The catalyzed reactions all followed Michaelis-Menten kinetics, and an impressive rate increase of up to 7569 (kcat/kuncat) was found for the hydroxycoumarin glucoside substrate 4-MUGP. Good and moderate degrees of catalysis (kcat/kuncat) of up to 1259 were found for the natural glucosides phloridzin and skimmin. By using a newly developed catechol detection UV-assay, a weak degree of catalysis was also found for the toxic hydroxycoumarin esculin. A novel synthesized diaminomethyl ß-cyclodextrin showed a weak catalysis of p-nitrophenyl ß-D-glucopyranoside hydrolysis.

Novel nojirimycin-derived bicyclic structures, containing cyclic carbamate, urea and guanidine moieties have been synthesised starting from suitably protected a-C-vinylnojirimycin and a-C-allylnojirimycin, respectively, and their biological activity against different glycosidases and as antibacterial agents tested

The transglycosylation activity of a novel alpha-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with alpha-(1-2), alpha-(1-4) and alpha-(1-6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one alpha-(1-6) linkage (panose, 6-O-alpha-glucosyl-maltotriose and 6-O-alpha-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the alpha-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial alpha-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.

Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work. Dextranase catalyzes the hydrolysis of α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49. Endo-β-1,4-D-mannanase catalyzes the depolymerization of β-1,4-mannan polymers. The structure of endo-1,4-β-mannanase Man5A from blue mussel Mytilus edulis has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium K-edge with selenomethionyl enzymes expressed in the yeast Pichia pastoris. Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the β-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from T. fusca in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted 2SO conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed 4C1 chair conformation.

Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work. Dextranase catalyzes the hydrolysis of α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49. Endo-β-1,4-D-mannanase catalyzes the depolymerization of β-1,4-mannan polymers. The structure of endo-1,4-β-mannanase Man5A from blue mussel Mytilus edulis has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium K-edge with selenomethionyl enzymes expressed in the yeast Pichia pastoris. Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the β-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from T. fusca in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted 2SO conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed 4C1 chair conformation.

Oligosaccharide processing enzymes such as glycosidases and glycosyltransferases are important classes of biocatalysts involved in synthesising specific oligosaccharide structures on proteins and lipids. These enzymes are known to be involved in a wide range of important biological processes, such as intestinal digestion, post-translational processing of glycoproteins, lysosomal catabolism of glycoconjugates and inter-cellular recognition events. Inhibition of these enzymes can disrupt biosynthesis of oligosaccharides, thus interfering in all of these processes. Hence, "glyco-enzyme" inhibitors might have enormous therapeutic potential in many diseases such as viral infection, cancer and diabetes. This very important prospect has led to increasing interest and demand for these compounds. Interference in oligosaccharide processing is the basis for the anti-influenza neuraminidase inhibitors that have recently been marketed and also for the potential use of glycosidase inhibitors against HIV, Gaucher's disease, hepatitis, and cancer. Since a rational design and synthesis of inhibitors are often extremely difficult due to the limited information regarding the structure of the active site, combinatorial approaches are particularly promising. This review will focus on synthetic efforts for the preparation of combinatorial libraries of glyco-enzyme inhibitors.

The three-dimensional (3D) structure of the catalytic domain of Gas1p, a protein belonging to the only family of beta-(1,3)-glucan transferases so far identified in yeasts and some pathogenic fungi (family GH-72), has been predicted by combining results derived from threading methods, multiple sequence alignments and secondary-structure predictions. The 3D model has allowed the identification of several residues that are predicted to play a crucial role in structural integrity, substrate recognition and catalysis. In particular, the model of the catalytic domain can be useful for designing site-directed mutagenesis experiments and for developing inhibitors of Gas1p enzymatic activity.

Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

QC 20141212

Brettanomyces bruxellensis and lactic acid bacteria are common microorganisms capable of modifying wine aroma and flavor. The activity of beta-glucosiclase against p-nitrophenyl-beta-D-glucopyranoside was determined in a model system for 14 strains of Brettanomyces bruxellensis yeast and 9 strains of lactic acid bacteria (Oenococcus oeni). All Brettanomyces strains and 7 Oenococcus strains exhibited enzymatic activity against this substrate. B. bruxellensis beta-glucosiclase activity was primarily intracellular; O. oeni showed some extracellular activity. Strains showing activity greater than 1000 nmole mL(-1) g dry cell mass(-1) 24 hr(-1) for Brettanomyces, or 100 nmole mL(-1) g dry cell mass(-1) 24 hr(-1) for Oenococcus, were evaluated for their effect on native Viognier grape glycosides. Neither genus was active on Viognier grape glycosides.

Brettanomyces bruxellensis and lactic acid bacteria are common microorganisms capable of modifying wine aroma and flavor. The activity of beta-glucosiclase against p-nitrophenyl-beta-D-glucopyranoside was determined in a model system for 14 strains of Brettanomyces bruxellensis yeast and 9 strains of lactic acid bacteria (Oenococcus oeni). All Brettanomyces strains and 7 Oenococcus strains exhibited enzymatic activity against this substrate. B. bruxellensis beta-glucosiclase activity was primarily intracellular; O. oeni showed some extracellular activity. Strains showing activity greater than 1000 nmole mL(-1) g dry cell mass(-1) 24 hr(-1) for Brettanomyces, or 100 nmole mL(-1) g dry cell mass(-1) 24 hr(-1) for Oenococcus, were evaluated for their effect on native Viognier grape glycosides. Neither genus was active on Viognier grape glycosides.

β1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, β1,3-glucan, and chitin. It has been postulated that the synthesis of β1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of β1,6- glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular β1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of β1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of β1,6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

Ricin is prototypical of many protein toxins, and is one of the most toxic compounds known to man. At the present time, no specific treatment is available for protein toxin exposure. Recent studies have shown that ricin exhibits a glycosidase activity which specifically removes an adenine base from rRNA, resulting in an inhibition of protein elongation and death of exposed animals. We have synthesized eight potential irreversible glycosidase inhibitors. The eight compounds were synthesized according to published methods, and the purities of the products were determined by melting point determination, elemental analysis, IR spectra, NMR spectra, and mass spectroscopy. Sufficient quantities of each of the eight compounds were synthesized to test their chemoprotectant activity against ricin in two cell lines, namely, a macrophage J744A.1 cell line and a Chinese hamster ovary cell line. The release of lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) were assessed as parameters of cytotoxicity after treatment with ricin or potential chemoprotectants. Alamine aminotransferase (ALT) was shown not to be a useful assay of cytotoxicity.