Search

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2′-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids. [ABSTRACT FROM AUTHOR]

Regular intake of fucosylated oligosaccharides has been associated with several benefits for human health, particularly for new-borns. Since these biologically active molecules can be found naturally in human milk, research efforts have been focused on the alternative synthetic routes leading to their production. In particular, utilization of fucosidases to perform stereoselective transglycosylation reactions has been widely investigated. Other reasons that bring these enzymes to the spotlight are their role in viral infections and cancer proliferation. Since their involvement in the pathogenesis of these diseases have been widely described, fucosidases have become a target in newly developed therapies. Finally, activity disorders of biologically important fucosidases can lead to health problems such as fucosidosis. What is common for both mechanisms is the interaction between the enzyme and substrates in and around the active site. Therefore, this review will analyse different substrate structures that have been tested in terms of their interaction with fucosidases active sites, either in synthesis or inhibition reactions. The published results will be compared from this perspective. [ABSTRACT FROM AUTHOR]

Context • Hepatitis B can develop into cirrhosis, and most liver cancers evolve on the basis of chronic hepatitis and cirrhosis. Many patients are already at an advanced stage when diagnosed. In recent years, clinicians have advocated detection of liver cancer using multiple markers in combination to improve the sensitivity and specificity of testing. Objective • The study aimed to evaluate the clinical value of using four tumor indicators—urea, alpha L-fucosidase (AFU), carbohydrate antigen 153 (CA153), carbohydrate antigen 125 (CA125), and alpha fetoprotein (AFP) and comparing the use of combined indicators to use of a single indicator for the diagnosis of liver cancer. Design • The research team performed a prospective study. Setting • The study took place at Clinical Laboratory, Baoding People’s Hospital, Baoding City, Hebei Province, China. Participants • Participants were 98 patients with chronic hepatitis B, who became the CHB group; 102 patients with liver cirrhosis, who became the cirrhosis group, and 100 patients with liver cancer, who became the liver cancer group. They all had been admitted to the hospital between March 2019 and March 2021. Outcome Measures • The research team measured the urea, AFU, CA153, CA125, and AFP levels of the three groups, constructed an ROC curve, and analyzed the diagnostic values of the indicators singly and in combination for liver cancer. Results • For the levels of urea, AFU, CA153, CA125, and AFP, the CHB group’s levels were significantly lower than those of the cirrhosis and liver cancer groups (both P < .001), and the cirrhosis group’s levels were significantly lower than those of the liver cancer group (P < .001). In the CHB group, the compensatory group’s levels were significantly lower than those of the decompensated group (P < .05). In the cirrhosis group, no significant differences existed between the levels of the grade A and grade B groups (P < .001), between those of the grade A and grade C groups (P < .001), or between those of the grade B and grade C groups (P < .001). In the cirrhosis group, the levels of the no ascites group were significantly lower than those of the ascites group (P < .05). In the liver cancer group, the levels of the stage I-II group were significantly lower than those of the stage III and stage IV groups (both P < .05), and those of the stage III group were significantly lower than those of the stage IV group (P < .05). The levels of the <5cm group were significantly lower than those of the ≥5cm group (P < .001). The value of using a combination of indicators for diagnosis was significantly higher than that of a single indicator (P < .001). Conclusions • Urea, AFU, CA153, CA125, and AFP all have diagnostic value in the evaluation of chronic hepatitis B-cirrhosis and liver cancer, with the highest efficacy, sensitivity and specificity from a combined test and diagnosis. [ABSTRACT FROM AUTHOR]

Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre‐sized droplets allow the very efficient use of protein samples and can produce large quantities of high‐quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X‐ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample‐transport and data‐collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50–500 pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data‐collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X‐ray beam by depositing an emulsion of droplets onto a silicon fixed‐target serial device are provided. After three months of storage at 4°C, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample‐delivery and data‐collection strategy allows crystal droplets to be shipped and set aside until beamtime is available. [ABSTRACT FROM AUTHOR]

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 a-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 a-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific a-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. [ABSTRACT FROM AUTHOR]

Human milk is the optimal nutrition source for infants, and oligosaccharides represent the third most abundant component in milk after lactose and fat. Human milk oligosaccharides (HMO) are favorable macromolecules which are, interestingly, indigestible by the infant but serve as substrates for bacteria. Hypothesizing that the maternal diet itself might influence HMO composition, we sought to directly determine the effect maternal diet on HMO and the milk bacteria. Employing a human cross-over study design, we demonstrate that distinct maternal dietary carbohydrate and energy sources preferentially alter milk concentrations of HMO, including fucosylated species. We find significant associations between the concentration of HMO-bound fucose and the abundance of fucosidase (a bacterial gene that digests fucose moieties) harbored by milk bacteria. These studies reveal a successive mechanism by which the maternal diet during lactation alters milk HMO composition, which in turn shapes the functional milk microbiome prior to infant ingestion. [ABSTRACT FROM AUTHOR]

Fucosyl-N-acetylglucosamine disaccharides are present in many biologically important oligosaccharides, such as human milk oligosaccharides, Lewis carbohydrate antigens, and glycans on cell-surface glycoconjugate receptors, and thus have vast potential for infant formulas, prebiotics, and pharmaceutical applications. In this work, in order to screen biocatalysts for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides, we performed sequence analysis of 12 putative and one known α-L-fucosidases of Bacteroides fragilis NCTC9343 and constructed a phylogenetic tree of the nine GH29 α-L-fucosidases. After that, five GH29A α-L-fucosidases were cloned, and four of them were successfully heterogeneous expressed and screened for transglycosylation activity, and a GH29A α-L-fucosidase (BF3242) that synthesized a mix of Fuc-α-1,3/1,6-GlcNAc disaccharides using pNPαFuc as donor and GlcNAc as acceptor was characterized. The effects of initial substrate concentration, pH, temperature, and reaction time on its transglycosylation activity were studied in detail. Under the optimum conditions of 0.05 U/mL enzyme, 20 mM pNPαFuc, and 500 mM GlcNAc in sodium buffer (pH 7.5) at 37 °C for 45 min, BF3242 efficiently synthesized Fuc-α-1,3/1,6-GlcNAc at a maximum yield of 79.0% with the ratio of 0.48 for 1,3/1,6. The molecular dynamics simulation analysis revealed that Loop-4 (His220-Ser245) in the putative 3D model of BF3242 displayed significant changes throughout the thermal simulations, might being responsible for the changes in the ratio of two regioisomeric products at different temperatures. This work provided not only a potential synthetic tool for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides but also a possibility for the formation of regioisomeric products in glycosidase-catalyzed transglycosylation. Key points: • Sequence analysis of α-L-fucosidases of Bacteroides fragilis NCTC9343 • Obtainment of an α-L-fucosidase with high transglycosylation activity • Explanation why temperature affected the ratio of two regioisomeric products [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthesis of N -alkylated 1-deoxyfuconojirimycins as α-fucosidase inhibitors. • N -Decyl-1-deoxyfuconojirimycin was toxic towards various cancer cells. • Development of homology models for fucosidases from bovine and human origin. Abstract 1,5-Dideoxy-1,5-imino- l -fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N -alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B. taurus (bovine origin). The N -alkyl derivatives were inactive against the bacterial fucosidase, while inhibiting the bovine enzyme. Docking of inhibitors to homology models, generated for the bovine and human fucosidases, was carried out. N -Decyl-DFJ was toxic to cancer cell lines and was more potent than the other N -alkyl DFJs studied. [ABSTRACT FROM AUTHOR]

α-L-Fucosidase (AFU) is a promising therapeutic target for the treatment of inflammation, cancer, cystic fibrosis, and fucosidosis. Some of the existing analytical methods for the assessment of AFU activity are lacking in sensitivity and selectivity, since most of them are based on spectrofluorimetric methods. More recently, mass spectrometry (MS) has evolved as a key technology for enzyme assays and inhibitor screening as it enables accurate monitoring of the conversion of substrate to product in enzymatic reactions. In this study, UHPLC-MS has been utilized to develop a simple, sensitive, and accurate assay for enzyme kinetics and inhibition studies of AFU3, a member of the AFU family. A reported method for analyzing saccharide involving a porous graphitic carbon column, combined with reduction by NaBH4/CH3OH, was used to improve sensitivity. The conversion of saccharide into alditol could reach nearly 100% in the NaBH4 reduction reaction. In addition, the bioanalytical quantitative screening method was validated according to US-FDA guidance, including selectivity, linearity, precision, accuracy, stability, and matrix effect. The developed method displayed a good accuracy, high sensitivity (LOD = 0.05 mg L−1), and good reproducibility (RSD < 15%). The assay accurately measured an IC50 value of 0.40 μM for the known AFU inhibitor, deoxyfuconojirimycin, which was consistent with results reported in the literature. Further validation of the assay was achieved through the determination of a high Z′-factor value of 0.89. The assay was applied to screen a marine-derived chemical library against AFU3, which revealed two marine-oriented pyrimidine alkaloids as potential AFU3 inhibitors. Graphical abstract [ABSTRACT FROM AUTHOR]

Fucosylated oligosaccharides play important physiological roles in humans, including in the immune response, transduction of signals, early embryogenesis and development, growth regulation, apoptosis, pathogen adhesion, and so on. Efforts have been made to synthesize fucosylated oligosaccharides, as it is difficult to purify them from their natural sources, such as human milk, epithelial tissue, blood, and so on. Within the strategies for its in vitro synthesis, it is remarkable the employment of fucosidases, enzymes that normally cleave the fucosyl residue from the non‐reducing end of fucosylated compounds, as these enzymes are also capable of synthesizing them by means of a transfucosylation reaction. This review summarizes the progress in the use of fucosidases for the synthesis of compounds that have potential for industrial and commercial applications. [ABSTRACT FROM AUTHOR]

α- l -Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l -fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides. [ABSTRACT FROM AUTHOR]

Objectives: Investigation of long-term dynamic changes of salivary activity/output of exoglycosidases, deglycosylation processes and their applicability as alcohol markers. Methods: Exoglycosidase (α-fucosidase (FUC), β-galactosidase (GAL), β-glucuronidase (GLU), β-hexosaminidase (HEX, HEX A and HEX B isoenzymes) and α-mannosidase (MAN)) activities were measured in the saliva of healthy social drinking controls (C), alcohol-dependent non-smokers (ANS) and alcohol-dependent smokers (AS) at the 1st, 15th, 30th and 50th day of abstinence after chronic alcohol drinking. Results: The activity of exoglycosidases was 2-3-fold (MAN), 2-6 fold (FUC), 8-25-fold (HEX A) and 19-40-fold (GLU) higher in the ANS and AS groups than in controls, and had good/excellent sensitivity, specificity and accuracy. The higher outputs of exoglycosidases were in the AS and ANS groups than in controls at the 1st day (GLU, HEX A) and at the 50th day (GLU, FUC, MAN) of abstinence. We found numerous correlations between alcohol-drinking days with GLU and HEX A, alcohol amounts with HEX A and duration of alcohol dependence with FUC and MAN activity/output. Conclusions: Salivary exoglycosidases/deglycosylation processes were still very high up to 50 days after the end of alcohol consumption. We found markers of chronic alcohol consumption (HEX A), alcohol dependence (FUC and MAN) and chronic alcohol consumption and dependence (GLU). [ABSTRACT FROM AUTHOR]

Abstract Hepatocellular carcinoma (HCC) is among the leading causes of mortality in the world. The detection of HCC in its early stage is the key for early treatment and thus the improvement of the chances of survival. Among the various methods of HCC screening, assays based on the detection of biomarker that is specific to HCC such as alpha- l -fucosidase (AFU) have been regarded as the most prominent methods. In this regards, a new assay for the detection of AFU to screen HCC was developed. This assay was based on the energy transfer between carbon dots (C-dots) and gold nanoparticles (AuNPs), the concentration of AFU could be monitored by the degree of C-dots fluorescence quenching due to the energy transfer. With this assay, a limit of detection of 3.4 nM (well below the diagnostic cutoff point of 80 nM), and a broad linear range of detection from 11.3 to 200 nM were achieved. We also demonstrate the determination of the concentration of AFU in human blood serum. Graphical abstract Image 1 Highlights • Energy transfer between carbon dots and AuNPs was used to design assay for the detection of alpha- l -fucosidase. • A sandwich assay was designed by combining polyclonal antibody coated AuNPs with monoclonal antibody attached to carbon dots. • A limit of detection of 3.4 nM and linear range from 11.3 to 200 nM were achieved. [ABSTRACT FROM AUTHOR]

α- l -Fucosidases are widely occurring enzymes that remove fucose residues from N - and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α- l -fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α- l -fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α- l -fucosidase from the spider Nephilingis cruentata (NcFuc) with a K mvalue for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto- N -difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower k catof D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower k catvalue of E59A. This was supported by the 57-fold increase in the k catof E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases. [ABSTRACT FROM AUTHOR]

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-L-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans. [ABSTRACT FROM AUTHOR]

The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides. [ABSTRACT FROM AUTHOR]

Abstract Purpose Adaptation of the colorimetric method for the determination of β- d -galactosidase, β- d -glucuronidase and α- l -fucosidase activities in serums from hemolyzed blood, the material currently being discarded. Materials and Methods The materials included serums from hemolyzed and non-hemolyzed blood, obtained from 26 healthy volunteers. The adaptation of the method involved precipitation of the proteins with trichloroacetic acid after incubating serums with substrates, but before determining the products of enzymatic reactions. Results In serums from hemolyzed and non-hemolyzed blood of the same persons, we found high correlations among the results obtained using hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods. Conclusion We are able to determine the β- d -galactosidase, β- d -glucuronidase and α- l -fucosidase activities in serums from hemolyzed blood (with adapted) and non-hemolyzed blood (with non-adapted) methods, with the same accuracy and precision. [ABSTRACT FROM AUTHOR]

Abstract Purpose The purpose of the study was to determine the effect of age on lysosomal exoglycosidase activities: α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase in healthy children and adolescents. Material and methods Urine samples were collected from 203 healthy children and adolescents (girls = 99, boys = 104), aged six months to 17.9 years. The activities of α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase were determined by colorimetric method and expressed in pKat/μg of creatine (pKat/μg Cr.). Results Urinary α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase activities (pKat/μg Cr.) were the highest in children below 3 years of age in comparison to the remaining age groups. There was a statistically significant negative correlation between urinary α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase (pKat/μg Cr.) and age (r = −0.36; r = −0.36; r = −0.35; r = −0.35; at p < 0.0001, respectively). In addition, we constructed the reference values for urinary activity of α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase (pKat/μg Cr.) in percentiles according to age in 3-year intervals. Conclusions Our study is the first to show reference values for urinary α-fucosidase, β-galactosidase, β-glucuronidase and α-mannosidase in children and adolescents. [ABSTRACT FROM AUTHOR]

The α-1,3/4- l -fucosidases (EC 3.2.1.111; GH29) Bb AfcB from Bifidobacterium bifidum and Cp Afc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto- N -fucopentaose II (LNFP II) through regioselective transfucosylation of lacto- N- tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering Bb AfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of Bb AfcB with the corresponding 17-aminoacid α-helical loop of Cp Afc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT Bb AfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4- l -fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT Bb AfcB (14 ± 0.3%) and comparable to that obtained with Cp Afc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto- N -neotetraose (LNnT) could be enriched with LNFP III. [ABSTRACT FROM AUTHOR]

Synthetic analogues of the naturally occurring iminosugar homoDMDP, which feature a perfluoroalkyl group at the pseudo-anomeric position, have been synthesized from the corresponding sugar-derived cyclic aldonitrone. The new fluorinated iminosugars as well as homoDMDP and its 6-deoxy counterpart were evaluated for their inhibitory activity against a panel of glycosidases. While the replacement of the (1′,2′)-dihydroxyethyl substituent of homoDMDP with –C 4 F 9 proved detrimental for enzyme binding, introduction of a –C 3 F 7 moiety tuned the inhibitory activity spectrum selectively towards α-fucosidase and α-glucosidase from yeast. [ABSTRACT FROM AUTHOR]

In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1, 3/4-fucosidase (α-Fuc'ase Sl-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc'ase Sl-1 is involved in the turnover of Lea epitope-containing N-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1, 3/4-fucosidase (α-Fuc'ase Sl-2), which is also active toward the complex type N-glycans containing Lea epitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc'ase Sl-2 with anti-FLAG tag, and the expression product (rFuc'ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc'ase Sl-1, rFuc'ase Sl-2 hydrolyzed the non-reducing terminal α1, 3-fucose residue on LNFP III and α1, 4-fucose residues of Lea epitopes on plant complex type N-glycans, but not the core α1, 3-fucose residue on Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc'ases Sl-1 and Sl-2 were specifically active toward α1, 3-fucose residue on GlcNAcb1-4(Fucα1-3)GlcNAc, indicating that the nonsubstituted b-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specific N-glycans must be a pre-requisite for α-Fuc'ase activity. A 3D modelled structure of the catalytic sites of α-Fuc'ase Sl-2 suggested that Asp192 and Glu236 may be important for binding to the α1, 3/4 fucose residue. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α- l -fucosidases is a new route for manufacturing biomimetic HMOs. Seven α- l -fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α- l -1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto- N- fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto- N -tetraose as acceptor with a 39% yield. α- l -Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2′-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively. [ABSTRACT FROM AUTHOR]

All reported α-L-fucosidases catalyze the removal of nonreducing terminal L-fucoses from oligosaccharides or their conjugates, while having no capacity to hydrolyze core fucoses in glycoproteins directly. Here, we identified an α-fucosidase from the bacterium Elizabethkingia meningoseptica with catalytic activity against core α-1,3-fucosylated substrates, and we named it core fucosidase I (cFase I). Using site-specific mutational analysis, we found that three acidic residues (Asp-242, Glu-302, and Glu-315) in the predicted active pocket are critical for cFase I activity, with Asp-242 and Glu-315 acting as a pair of classic nucleophile and acid/base residues and Glu-302 acting in an as yet undefined role. These findings suggest a catalytic mechanism for cFase I that is different from known α-fucosidase catalytic models. In summary, cFase I exhibits glycosidase activity that removes core α-1,3-fucoses from substrates, suggesting cFase I as a new tool for glycobiology, especially for studies of proteins with core fucosylation. [ABSTRACT FROM AUTHOR]

Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and β1,2-linked xylose towards the β- mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex-type N-glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N-glycans. We found that this glycoside hydrolase specifically acted on GlcNAcβ1-4(Fucα1-3)GlcNAc, a degradation product of plant complex-type N-glycans, by sequential actions of vacuolar α-mannosidase, β1,2-xylosidase, and endo-β-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcβ1-4 (Fucα1-3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3- fucosidase acting on plant complex-type N-glycans and elucidated the degradation pathway of plant complex-type N-glycans. [ABSTRACT FROM AUTHOR]

The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I. = 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I. = 540.5 ± 49.93) (P < 0.05). This decrease was in line with the increased activity of α- l -fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I. = 1.45 ± 0.08 to 1.06 ± 0.03) (P < 0.05) indicating that the α- l -fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α- l -fucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis. [ABSTRACT FROM AUTHOR]

Fucosidosis is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-L-fucosidase. We present the case of an affected female in the second decade of life with chronic rhinosinusitis (CRS) including recalcitrant polypoid inflammation, which has not been previously reported in the literature. With the advancement of life-prolonging measures, children with lysosomal storage disorders may suffer increasingly from CRS due to the lymphohistiocytic and macrophage infiltrate of the paranasal sinus mucosa that resembles severe polypoid inflammation. [ABSTRACT FROM AUTHOR]

Detection of alpha-l-fucosidase has been shown to have relevance in diagnosing hepatocellular carcinoma. Few assays have been developed to measure this enzyme, with most relying on colorimetric techniques involving the enzyme's kinetics. While these assays are facile and quick, the sensitivity is not always sufficient for early tumor detection. To improve upon previous assays for alpha-l-fucosidase, a fluorescence based immunoassay was produced implementing an alpha-l-fucosidase specific antibody (FUCA2). The immobilization of the alpha-l-fucosidase-specific antibody onto a quartz slide was investigated with several bioconjugation approaches and an immunoassay for detection of alpha-l-fucosidase was produced. The immunoassay was utilized to produce calibration curves for quantifying alpha-l-fucosidase concentrations in both PBS and human blood serum. A detection limit of 10 nM was found using human blood serum, which is well below the diagnostic cutoff point of 80 nM. [ABSTRACT FROM AUTHOR]

The native Putranjiva roxburghii family 1 glycoside hydrolase enzyme showed β-D-fucosidase activity in addition to β-D-glucosidase and β-D-galactosidase activities reported in our previous study. A single step concanvalin A affinity chromatography for native PRGH1 improved the yield and reduced the purification time. The PRGH1 gene was cloned and overexpressed in E. coli . The full length gene contained an ORF of 1617 bp encoding a polypeptide of 538 amino acids. The amino acid sequence of PRGH1 showed maximum similarities to β-glucosidases and myrosinases. Both native and recombinant protein showed maximum hydrolytic activity for p NP-Fuc followed by p NP-Glc and p NP-Gal. Significant enzyme activity was also observed for cellobiose, however it decreased with increase in chain-length for glycan substrates. The enzyme showed significant resistant to D-glucose concentration up to 500 mM. Mutational studies confirmed the predicted catalytic acid/base Glu173 and nucleophile Glu389 as key residues for its activity. Moreover, Glu446 and Asn172 played essential role in substrate binding by interacting with the −1 subsite of substrates. Bioinformatic analysis suggested the possible reasons for the broad substrate specificity and other properties of the enzyme. PRGH1 had high sequence similarity towards S-glucosidase and may be involved in defence. The broad specificity, catalytic efficiency and thermostability make PRGH1 potentially an important industrial enzyme. [ABSTRACT FROM AUTHOR]

In this study, we identified α gene in tomαto thαt encodes αn αcidic α-fucosidαse (LOC101254568 or Solyc03g006980, α-Fuc'αse S1-1), which mαy βe involved in the turnover of plαnt complex-type N-glycαns. Recomβinαnt α-Fuc'αse S1-1 (rFuc'αse S1-1) wαs expressed using α βαculovirus_insect cell expression system. rFuc'αse Sl-1 is 55 kDα in size αnd hαs αn optimum pH αround 4.5. It suβstαntiαlly hydrolyzed the non-reducing terminαl α1,3-fucose residue on LNFP III αnd α1,4-fucose residues of Leα epitopes on plαnt complex-type N-glycαns, βut not the α1,2-fucose residue on LNFP I or the α1,3-fucose residue on pyridylαminαted Fucα1-3GlcNAc. Furthermore, we found thαt this tomαto α-Fuc'αse S1-1 wαs inαctive towαrd the core pentα-oligosαcchαride unit [Mαnβ1-4(Xylβ1-2)GlcNAcβ1-4(Fucα1-3)GlcNAc-PA] of plαnt complex-type N-glycαns. Moleculαr 3D modelling of α-Fuc'αse Sl-1 αnd structure/sequence interpretαtion βαsed on compαrison with α homologous α-fucosidαse from Bifidoβαcterium longum suβsp. infαntis (Blon_2336) indicαted thαt residues Asp193 αnd Glu237 might βe importαnt for suβstrαte βinding. [ABSTRACT FROM AUTHOR]

α- l -Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α- l -Fucosidases, glycoside hydrolases for catalyzing the removal of α- l -fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α- l -fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α- l -fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α- l -fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α- l -fucosidases. [ABSTRACT FROM AUTHOR]

Here, we report the biochemical characterization of a novel α- l -fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α- l -fucosidase was obtained from several chromatographic steps after growth in the presence of l -fucose. The purified α- l -fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p -nitrophenyl α- l -fucopyranoside ( p NPFuc), with K m = 1.1 ± 0.1 mM and k cat = 39.8 ± 1.8 s −1 . l -fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited p NPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α- l -fucosidase that preferentially hydrolyzes the α-(1 → 4)- L -fucosidic linkage present in α- L -fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and p NP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds. [ABSTRACT FROM AUTHOR]

Alpha-l-fucosidase is a known biomarker for hepatocellular carcinoma that has shown great potential in diagnostics. Most of the focus for this enzyme has been on the free form found in serum; however, little is known of the properties of the minor portion of membrane-bound alpha-l-fucosidase. To better understand the properties of membrane-bound alpha-l-fucosidase, this enzyme was surveyed at the air-water interface. Alpha-l-fucosidase is able to form a stable Langmuir monolayer, which was confirmed through surface-pressure and surface-potential area isotherms, as well as infrared reflection-absorption spectroscopy (IRRAS). Furthermore, an interaction between the alpha-l-fucosidase Langmuir monolayer and a specific antibody for this enzyme, FUCA2, was observed. [ABSTRACT FROM AUTHOR]

Three novel bacterial a-L-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-a-L-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively a1,6-linked fucose and Eo3812 released only a1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca2+, Mn2+ and Zn2+ ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-a-L-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15%transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications. [ABSTRACT FROM AUTHOR]

In present work we provide the bioinformatic and biochemical characterization of six α-L-fucosidases that belong to the 29 and 95 families of glycoside hydrolases (GH) from the fucoidan-degrading locus of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The fucosidases FucWf1GH29, FucWf2GH29, FucWf3GH29 and FucWf6GH29 are relegated to the subfamily A of the GH29 family. The fucosidase FucWf4GH29 bears a distant resemblance to the GH29 and does not belong to either the GH29A or the GH29B subfamilies. Apparently, FucWf4GH29 is the first representative of a new subfamily within the GH29 family of α-L-fucosidases. For the first time the specificity of fucosidases has been studied using a series of fucoidan-related sulfated oligosaccharides. Studied α-L-fucosidases are able to cleave l -fucose from sulfated fucooligosacchrides after their treatment with exo-sulfatases. All studied α-L-fucosidases are cleaving the α-1→3- and α-1→4-linked terminal l -fucose in sulfated fucooligosaccharides. However, only FucWf3GH29 is able to cleave off an α-1→2-linked l -fucose. The fucosidase FucWf5GH95 of the GH95 family is shown to have higher activity on fucoidans than fucosidases of the GH29 family. Supposedly, the α- l -fucosidase FucWf5GH95 participates in fucoidan debranching. The obtained data indicate different roles of fucosidases of the GH29 and GH95 families in the process of fucoidan degradation by the marine bacteria W. fucanilytica CZ1127T. • Fucosidase FucWf4GH29 is a member of a new subfamily within the GH29 family. • Fucosidases FucWf1-5 cleave off l -fucose residues from sulfated oligosaccharides after their treatment with sulfatases. • Studied fucosidases are differing in specificity against the types of glycosidic bonds in fucooligosaccharides. • Studied fucosidases of the GH29 and GH95families shown different selectivity against fucooligosaccharides or fucoidans. • Fucosidase FucWf5GH95 is involved in debranching of the fucoidans. [ABSTRACT FROM AUTHOR]

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses. [ABSTRACT FROM AUTHOR]

α-Fucosidosis is a rare inherited neuro-degenerative disorder causing progressive neurological deterioration leading to early death. Definitive diagnosis requires α-fucosidase enzyme assay or FUCA1 gene testing, which being expensive limits the definitive diagnosis in resource limited countries. We present two siblings with classic symptoms, radiological and MRI brain findings suggestive of α-fucosidosis and a clinical approach to reach to the diagnosis. [ABSTRACT FROM AUTHOR]

Fucosidosis is a rare lysosomal storage disorder caused by deficient activity of the enzyme l -fucosidase in all tissues. We presented magnetic resonance imaging [MRI] and MR spectroscopy [MRS] findings of a 4-year-old boy with genetically proven fucosidosis. He had a history and clinical findings of recurrent sinopulmonary infections, hypertonicity on lower extremities, gingival hypertrophy, bilateral ptosis, angiokeratoma corporis diffusum, and dysostosis multiplex. He had no organomegaly and urine glycosaminoglycan analysis were normal. MRI revealed abnormalities within the globus pallidus and periventricular and subcortical white matter. MRS showed a peak at the 3.8–3.9 ppm as a result of accumulating carbohydrate containing macromolecules and another peak at 1.2 which was doublet and inverted on TE 135, suggesting fructose peak. A final diagnosis of fucosidosis was proved by mutational analysis of FUCA1 gene which is responsible for the Fucosidosis phenotype. Two recent reports of MRS of two patients demonstrated that MRS is specific for fucosidosis. In this case, we aim to discuss fucosidosis with MRI and MRS findings accompanied by the literature. [ABSTRACT FROM AUTHOR]

The synthesis and the biological evaluation of a new ferrocenyl-iminosugar conjugate designed for fucosidase inhibitory and anticancer activity is described. The compound showed strong affinity for fucosidase from bovine kidney ( K i = 23 nM) and from Bacteroides thetaiotaomicron ( K i = 150 nM), displaying a 10-fold tighter binding affinity for these enzymes than the previous analogs. The interaction pattern that improves binding has been evaluated through structural analysis of the inhibitor–enzyme complex. The ferrocenyl-iminosugar exhibits significant anticancer activity on MDA-MB-231 and SK-MEL28 cell lines at 100 μM. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-L-Fucosidases catalyze the hydrolysis of α-L-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-L-fucosidase from Thermotoga maritima (TmaFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-Ltransfucosidase starting with the α-L-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs. [ABSTRACT FROM AUTHOR]

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides. [ABSTRACT FROM AUTHOR]

We have established a concise synthetic route relying on a key base-promoted epimerization step to synthesize two series of activity-based probes carrying a BODIPY fluorophore for α-L-fucosidase. The resulting probes were evaluated for labeling performance. The one utilizing an o-fluoromethylphenol derivative as the latent trapping unit was successfully applied for the first time to visualize and locate lysosomal α-l-fucosidase activity in human cells. [ABSTRACT FROM AUTHOR]

Sperm–oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein–carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α- l -fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α- l -fucosidases have fundamental function(s) during gamete interactions. An α- l -fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila . Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster . In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α- l -fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α- l -fucosidase bound to fucose residue and in particular it prefers N -glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N -glycans carrying only a terminal mannose residue. The ability of sperm α- l -fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α- l -fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α- l -fucosidase was pre-incubated with fucoidan, a polymer of α- l -fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α- l -fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm–egg interactions in Drosophila . [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]

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 μM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase. [ABSTRACT FROM AUTHOR]

Fucosidase inhibitors represent captivating targets for various biological applications. Iminosugars featuring a fluoroalkyl chain in the pseudoanomeric position were synthesized by nucleophilic addition of a perfluoroalkyl Grignard reagent to a nitrone. Reduction of the N-O bond of the hydroxylamine was achieved by treatment with an aqueous solution of sulfur dioxide. The p Ka = 4.5 of the target pyrrolidine reflected a strong electronic effect of the fluoroalkyl chain. Inhibitory potencies of the fluorinated iminosugars and their corresponding hydroxylamines were evaluated against α- L-fucosidase at various pH values. [ABSTRACT FROM AUTHOR]

The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (1VF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa-ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were coincubated for 15 min. A significant increase in spermatozoa-ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa. [ABSTRACT FROM AUTHOR]

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni . Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, α-glucosidase, trehalase, and α-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of α-L-fucosidase. The isolated α-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata α-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented K m values of 48.4 μM to 4-Methylumbelliferyl α-L-fucopyranoside and 0.55 mM to p-nitrophenyl-α-L-fucopyranoside. Thus, α-L-fucosidase has a high activity in the hepatopancreas of B. glabrata , and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, α-L-fucosidase may also be important in host/parasite interactions. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel marine-derived α- l -fucosidase OUC-Jdch16 was cloned and characterized. • The OUC-Jdch16 was applied to produce 2′-FL with the highest yield of 92.15%. • Other novel glycosides were prepared via the OUC-Jdch16. 2′-Fucosyllactose (2′-FL) is one of the nutrient ingredients in human milk, which has various beneficial health effects. α- l -fucosidase is a biotechnological tool for 2′-FL preparation. Here, a novel and efficient α- l -fucosidase OUC-Jdch16 from the fucoidan-digesting strain Flavobacterium algicola 12076 was heterologously expressed and applied to produce 2′-FL in vitro. OUC-Jdch16 belongs to glycoside hydrolases (GH) family 29 and exhibits the highest 4-nitrophenyl-α- l -fucopyranoside-hydrolyzing activity at 25 °C and pH 6.0. OUC-Jdch16 could catalyze the synthesis of 2′-FL via transferring the fucosyl residue from pNP-α-fucose to lactose. Under the optimal transfucosylation conditions, the yield of the transfucosylation product reached 84.82% and 92.15% (mol/mol) from pNP-α-fucose within 48 h and 120 h, respectively. Moreover, OUC-Jdch16 was capable of transferring the fucosyl residue to other glycosyl receptors with the generation of novel fucosylated compounds. This study demonstrated that OUC-Jdch16 could be a promising tool to prepare 2′-FL and other novel glycosides. [ABSTRACT FROM AUTHOR]

Background Mixed microbial cultures, in which bacteria and fungi interact, have been proposed as an efficient way to deconstruct plant waste. The characterization of specific microbial consortia could be the starting point for novel biotechnological applications related to the efficient conversion of lignocellulose to cello-oligosaccharides, plastics and/or biofuels. Here, the diversity, composition and predicted functional profiles of novel bacterial-fungal consortia are reported, on the basis of replicated aerobic wheat straw enrichment cultures. Results In order to set up biodegradative microcosms, microbial communities were retrieved from a forest soil and introduced into a mineral salt medium containing 1% of (un)treated wheat straw. Following each incubation step, sequential transfers were carried out using 1 to 1,000 dilutions. The microbial source next to three sequential batch cultures (transfers 1, 3 and 10) were analyzed by bacterial 16S rRNA gene and fungal ITS1 pyrosequencing. Faith's phylogenetic diversity values became progressively smaller from the inoculum to the sequential batch cultures. Moreover, increases in the relative abundances of Enterobacteriales, Pseudomonadales, Flavobacteriales and Sphingobacteriales were noted along the enrichment process. Operational taxonomic units affiliated with Acinetobacter johnsonii, Pseudomonas putida and Sphingobacterium faecium were abundant and the underlying strains were successfully isolated. Interestingly, Klebsiella variicola (OTU1062) was found to dominate in both consortia, whereas K. variicola-affiliated strains retrieved from untreated wheat straw consortia showed endoglucanase/xylanase activities. Among the fungal players with high biotechnological relevance, we recovered members of the genera Penicillium, Acremonium, Coniochaeta and Trichosporon. Remarkably, the presence of peroxidases, alpha-L-fucosidases, beta-xylosidases, beta-mannases and beta-glucosidases, involved in lignocellulose degradation, was indicated by predictive bacterial metagenome reconstruction. Reassuringly, tests for specific (hemi)cellulolytic enzymatic activities, performed on the consortial secretomes, confirmed the presence of such gene functions. Conclusion In an in-depth characterization of two wheat straw degrading microbial consortia, we revealed the enrichment and selection of specific bacterial and fungal taxa that were presumably involved in (hemi)cellulose degradation. Interestingly, the microbial community composition was strongly influenced by the wheat straw pretreatment. Finally, the functional bacterial-metagenome prediction and the evaluation of enzymatic activities (at the consortial secretomes) revealed the presence and enrichment of proteins involved in the deconstruction of plant biomass. [ABSTRACT FROM AUTHOR]

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]