α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential.

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.

Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

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 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-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 α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Fucoidan-active α-L-fucosidases of the GH29 and GH95 families from a fucoidan degrading cluster of the marine bacterium Wenyingzhuangia fucanilytica.

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.

Architecture Insight of Bifidobacterial α-L-Fucosidases.

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics.

Screening and characterization of an α-L-fucosidase from Bacteroides fragilis NCTC9343 for synthesis of fucosyl-N-acetylglucosamine disaccharides.

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.

Integrative Proteomic Analysis of Digestive Tract Glycosidases from the Invasive Golden Apple Snail, Pomacea canaliculata.

The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata. A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

Synthetic and Crystallographic Insight into Exploiting sp2 Hybridization in the Development of α-l-Fucosidase Inhibitors.

The sugar fucose plays a myriad of roles in biological recognition. Enzymes hydrolyzing fucose from glycoconjugates, α-l-fucosidases, are important targets for inhibitor and probe development. Here we describe the synthesis and evaluation of novel α-l-fucosidase inhibitors, with X-ray crystallographic analysis using an α-l-fucosidase from Bacteroides thetaiotamicron helping to lay a foundation for future development of inhibitors for this important enzyme class.

Employment of fucosidases for the synthesis of fucosylated oligosaccharides with biological potential.

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.

Exploiting the hydrophobic terrain in fucosidases with aryl-substituted pyrrolidine iminosugars.

Fucosidase inhibition shows potential in numerous therapeutic contexts. Substitution of fucose-like iminosugars with hydrophobic "aglycons" yields significant improvements in potency of fucosidase inhibition. Here we have prepared three new 2-aryl-3,4-dihydroxy-5-methylpyrrolidines featuring phenyl substituents in variable orientations with respect to the iminocyclitol core and at various distances from it to explore the key binding interactions that stabilise the enzyme-inhibitor complex. The presence of a triazole linker in one structure resulted in nanomolar inhibition of the fucosidase from bovine kidney (Ki =4.8 nM), thus giving rise to one of the most potent pyrrolidine-type inhibitors of this enzyme known to date.

Community dynamics and metagenomic analyses reveal Bacteroidota's role in widespread enzymatic Fucus vesiculosus cell wall degradation.

Enzymatic degradation of algae cell wall carbohydrates by microorganisms is under increasing investigation as marine organic matter gains more value as a sustainable resource. The fate of carbon in the marine ecosystem is in part driven by these degradation processes. In this study, we observe the microbiome dynamics of the macroalga Fucus vesiculosus in 25-day-enrichment cultures resulting in partial degradation of the brown algae. Microbial community analyses revealed the phylum Pseudomonadota as the main bacterial fraction dominated by the genera Marinomonas and Vibrio. More importantly, a metagenome-based Hidden Markov model for specific glycosyl hydrolyses and sulphatases identified Bacteroidota as the phylum with the highest potential for cell wall degradation, contrary to their low abundance. For experimental verification, we cloned, expressed, and biochemically characterised two α-L-fucosidases, FUJM18 and FUJM20. While protein structure predictions suggest the highest similarity to a Bacillota origin, protein-protein blasts solely showed weak similarities to defined Bacteroidota proteins. Both enzymes were remarkably active at elevated temperatures and are the basis for a potential synthetic enzyme cocktail for large-scale algal destruction.

Structure and function of microbial α-l-fucosidases: a mini review.

Fucose is a monosaccharide commonly found in mammalian, insect, microbial and plant glycans. The removal of terminal α-l-fucosyl residues from oligosaccharides and glycoconjugates is catalysed by α-l-fucosidases. To date, glycoside hydrolases (GHs) with exo-fucosidase activity on α-l-fucosylated substrates (EC 3.2.1.51, EC 3.2.1.-) have been reported in the GH29, GH95, GH139, GH141 and GH151 families of the Carbohydrate Active Enzymes (CAZy) database. Microbes generally encode several fucosidases in their genomes, often from more than one GH family, reflecting the high diversity of naturally occuring fucosylated structures they encounter. Functionally characterised microbial α-l-fucosidases have been shown to act on a range of substrates with α-1,2, α-1,3, α-1,4 or α-1,6 fucosylated linkages depending on the GH family and microorganism. Fucosidases show a modular organisation with catalytic domains of GH29 and GH151 displaying a (ß/α)8-barrel fold while GH95 and GH141 show a (α/α)6 barrel and parallel ß-helix fold, respectively. A number of crystal structures have been solved in complex with ligands, providing structural basis for their substrate specificity. Fucosidases can also be used in transglycosylation reactions to synthesise oligosaccharides. This mini review provides an overview of the enzymatic and structural properties of microbial α-l-fucosidases and some insights into their biological function and biotechnological applications.

The dual role of fucosidases: tool or target.

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.

Discovery and characterization of a novel α-l-fucosidase from the marine-derived Flavobacterium algicola and its application in 2'-fucosyllactose production.

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.

Data collection from crystals grown in microfluidic droplets.

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.

The bone-degrading enzyme machinery: From multi-component understanding to the treatment of residues from the meat industry.

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.

Metataxonomic profiling and prediction of functional behaviour of wheat straw degrading microbial consortia.

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.