L-Fucose Assay Kit

Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.

Plant pathogenic fungi release cell wall degrading enzymes (CWDEs), which are significant weapons for breaking down plant cell walls, although only a few reports focus on their pathogenesis. The current study demonstrates that MoFco1, a conserved α-L-fucosidase in several pathogenic fungi, degrades the hemicellulose component XXFG and contributes to the pathogenicity of Magnaporthe oryzae. In addition, MoFco1 enzyme activity is essential for its pathogenic function, as the enzyme activity mutation induced pathogenesis defects identical to the ΔMofco1 mutant. We further performed a structure-based virtual screening targeting MoFco1 and discovered 0989, which binds to MoFco1 and effectively inhibits M. oryzae pathogenesis. In brief, our study revealed the pathogenic mechanism of α-L-fucosidase and explored the application of structure-based virtual screening in plant protection.

Biological glycans mediate several physiological processes, thus altered glycosylation patterns can lead to different diseases such as autoimmune, infectious, chronic anti-inflammatory diseases, or even cancer. In fact, alterations in fucosylation in either N- or O-glycans are among the most frequent changes in glycosylation patterns associated with cancer. Therefore, elucidation of the role of glycoconjugate glycans is essential for understanding the development of pathologies where they are involved. In this sense glycosidases are excellent tools, since they catalyse the selective removal of sugar residues, allowing the evaluation of changes in their biological role due to glycan removal. This work describes the purification and characterization of a α-fucosidase from the fungus Dichostereum sordulentum 1488. It is a homodimer with a molecular weight of 214 kDa and optimum pH and temperature of 4.0 and 70 °C respectively. It has a K_M of 0.27 mM and V_Max of 3.3 μmoles PNP/min per mg for the substrate p-nitrophenyl-α-l-fucopyranoside, showing a substrate inhibition profile. It showed high specificity for the hydrolysis of fucose linked by α-(1,2) bonds. The identification, purification, and characterization of this new α-fucosidase is highly relevant for enlarging the availability of glycosidases for use as tools for glycan elucidation.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid croplands competition between human food and animal feed. Agricultural co-products such as soybean meals have become important components of the circular economy thanks to their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet of monogastric animals, especially fungal carbohydrate-active enzymes (CAZymes). Here, we describe a time-course production and analysis of Aspergillus terreus secretomes for the identification of CAZymes able to enhance the digestibility of soybean meals. Functional assays revealed that the release of nutrients and the degradation of pectins in soybean meals can be tightly interconnected. Using a comparative proteomics approach, we identified several fungal pectin-degrading enzymes leading to increased assimilable nutrients in the soluble fraction of soybean meals. Our results reinforce the importance of deconstructing pectic polysaccharides in feedstuffs and contribute to sharpen our understanding of the fungal enzymatic interplays involved in pectin hydrolysis.IMPORTANCEIn the present study, we developed a strategy to identify the key fungal enzymatic activities involved in the improvement of soybean meal (SBM) digestibility. Our data unravel the importance of pectin degradation for the release of nutrients from SBM and provide some insights regarding the degradation of rhamnogalacturonan-I (RG-I) by ascomycetes. Indeed, the hydrolysis of pectins and RG-I by human microbiota is well documented in the literature, but our knowledge of the fungal CAZymes at play for the degradation of soybean pectins remains hitherto underexplored. Due to its wide use in animal feed, improving the digestibility of SBM by enzymatic treatments is a current challenge for feed additive suppliers. Since non-starch polysaccharides and pectins have often been reported for their anti-nutritional role in SBM, we believe this study will provide new avenues toward the improvement of enzymatic cocktails for animal nutrition and health.

Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages with diverse substrate/linkage specificities and can be used in transglycosylation reactions to synthesise oligosaccharides. Based on sequence identity, α-l-fucosidases have been classified in distinct glycoside hydrolases (GHs) families in the carbohydrate-active enzymes (CAZy) database. Here, we explored the sequence-function space of fucosidases from GH29 family. Based on sequence similarity network (SSN) analyses, 16 GH29 α-l-fucosidases were selected for functional characterisation. Using activity assays combined with HPAEC-PAD and LC-FD-MS/MS analyses, we determined the substrate and linkage specificities of these enzymes against a range of defined oligosaccharides and glycoconjugates, revealing a range of specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated ligands. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1-6 linkages and FA2G2 N-glycan was further determined by X-ray crystallography and saturation transfer difference NMR. TLC combined with electrospray ionization – MS and NMR confirmed the capacity of this enzyme to carry out transfucosylation reactions with GlcNAc and Fuc1,3GlcNAc as acceptors. Taken together, these experimental data validate the use of SSN as a reliable bioinformatics approach to predict the substrate specificity and transfucosylation activity of GH29 fucosidases.

Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α-L-fucosidase catalysis, in an effort toward drug design, has been hindered by the absence of three-dimensional structural data for any animal fucosidase. Here, we have used cryoelectron microscopy (cryo-EM) to determine the structure of human lysosomal α-L-fucosidase (FucA1) in both an unliganded state and in complex with the inhibitor deoxyfuconojirimycin. These structures, determined at 2.49 Å resolution, reveal the homotetrameric structure of FucA1, the architecture of the catalytic center, and the location of both natural population variations and disease-causing mutations. Furthermore, this work has conclusively identified the hitherto contentious identity of the catalytic acid/base as aspartate-276, representing a shift from both the canonical glutamate acid/base residue and a previously proposed glutamate residue. These findings have furthered our understanding of how FucA1 functions in both health and disease.

Bread is a high glycemic index (GI) food with high amounts of readily digestible carbohydrates. Fucoidan refers to a group of sulfated polysaccharides isolated from brown seaweed that has been gaining traction for its many functional properties, including its ability to inhibit starch hydrolases. In this study, fucoidan was added into bread to lower the glycemic index of bread. Fucoidan fortification at 3.0% reduced the starch digestion rate of baked bread by 21.5% as compared to control baked bread. This translated to a 17.7% reduction in the predicted GI (pGI) with 3.0% of fucoidan. Fucoidan was retained in the bread after baking. Although the in vitro bioavailability of fucoidan was negligible, the in vitro bioaccessibility of fucoidan was high, at 77.1–79.8%. This suggested that although fucoidan may not be absorbed via passive diffusion, there is potential for the fucoidan to be absorbed via other modes of absorption. Thus, there is a potential for the use of fucoidan as a functional ingredient in bread to reduce the glycemic potential of bread.

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.

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides’ (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

L-Fucose and L-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. L-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host–pathogen interactions. L-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated L-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L⁻¹ of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2′-fucosyllactose (2′FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2′FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2′FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2′FL from yeast, which is a key consideration for any biocatalyst for 2′FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2′FL efflux. Finally, we demonstrated production of 2′FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2′FL production.

2′-Fucosyllactose (2′-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2′-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2′-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and L-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni²⁺ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37°C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2′-FL in a large number of samples simultaneously. This method can be used to quantify 2′-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2′-FL.

Anthocyanins determine the color and potential health-promoting properties of red fruit juices, but the juices contain remarkably less anthocyanins than the fruits, which is partly caused by the interactions of anthocyanins with the residues of cell wall polysaccharides like pectin. In this study, pectin was modified by ultrasound and enzyme treatments to residues of polysaccharides and oligosaccharides widely differing in their molecular weight. Modifications decreased viscosity and degrees of acetylation and methylation and released smooth and hairy region fragments. Native and modified pectin induced different effects on the concentrations of individual anthocyanins after short-term and long-term incubation caused by both hydrophobic and hydrophilic interactions. Results indicate that both pectin and anthocyanin structure influence these interactions. Linear polymers generated by ultrasound formed insoluble anthocyanin complexes, whereas oligosaccharides produced by enzymes formed soluble complexes with protective properties. The structure of the anthocyanin aglycone apparently influenced interactions more than the sugar moiety.