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.

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.

The characterization of a recombinant glycosidase can be done with commercially available substrates, which enable testing of enzyme functionality and determination of linkage specificity. Colorimetric assays with p-nitrophenyl substrates provide a relatively simple and fast way of screening conditions which could affect enzyme activity (buffer, pH, ion dependence, temperature). These substrates are useful for the determination of activity optima and the characterization of basic activity parameters. However, testing for linkage specificity should be performed on more complex sugars presenting a range of different glycosidic bonds and might need more sophisticated methods of analysis. This protocol provides comprehensive instructions on how to perform an initial characterization of your glycosidase using a recombinant α-l-fucosidase as an example.

A diluted alkali-soluble pectin (DASP) fraction, extracted using sodium carbonate, is characterized by a low degree of methylesterification and has the ability to self-organize on mica. The aim of this study was to characterize the cross-linking process of this fraction, extracted from apples, over a wide pH range (3-11) and without the addition of salt. An FT-IR study showed an increase in the intensity of bands connected with ν_as and ν_s (COO⁻) and a decrease in the intensity of the band associated with ν (C=O) in the carboxyl group with increasing pH, which indicated the dissociation of the carboxyl groups of galacturonic acid units. An increase in the surface electrical charge of particles in the pH range of 3-7 confirmed this. The value of the average apparent dissociation constant (∼4.60) indicated the acidic character of the DASP fraction. An AFM study showed the morphological changes of the DASP fraction with increasing pH, which allowed for the evaluation of the cross-linking process. This fraction formed a network on mica at pH 4 and 9, while the aggregates were noted mainly at pH 11. For totally ionized carboxyl groups (pH 7), the pectin chains were separated from each other due to the electrostatic repulsion between them.

A highly regular fucan sulfate SHFS was isolated from the sea cucumber Stichopus horrens by extraction of the body walls in the presence of papain followed by ion-exchange and gel permeation chromatography. SHFS had MW of about 140 kDa and contained fucose and sulfate in the molar ratio of about 1:1. Chemical and NMR spectroscopic methods were applied for the structural characterization of the polysaccharide. SHFS was shown to have linear molecules built up of 3-linked α-L-fucopyranose 2-sulfate residues. Anticoagulant properties of SHFS were assessed in vitro in comparison with the LMW heparin (enoxaparin) and totally sulfated 3-linked α-L-fucan. SHFS was found to have the lowest activity, and hence, both sulfate groups at O-2 and O-4 of fucosyl units seem to be important for anticoagulant effect of sulfated homo-(1 → 3)-α-L-fucans.

The main nutritional/bioactive compounds (protein; aminoacids, AA; fucose; minerals; vitamins B12 and C; and total phenolic content, TPC) of nine commercial algae powders, used as food supplements, were studied. Undaria pinnatifida showed the highest protein/aminoacid contents (51.6/54.4 g 100 g^-1). Among brown macroalgae, Himanthalia elongate showed the highest fucose content (26.3 g kg^-1) followed by Laminaria ochroleuca (22.5 g kg^-1). Mineral contents of 15-24% were observed in the algae, being particularly excellent sources of iodine (69.0-472.0 mg kg^-1). Porphyra spp. and Palmaria palmata showed the highest vitamin B12 contents (667-674 µg kg^-1). Vitamin C ranged among 490.4-711.8 mg kg^-1. H. elongate showed the highest total phenolic content (14.0 g kg^-1). In conclusion, the studied algae are excellent sources of protein, AA, minerals, vitamin C and some of them presented particularly high vitamin B12 and fucose contents, which may have a potential use as food supplements.

Background: oral cancer is a result of disordered cellular behavior initiated by various stimuli which is characterized by the alteration of serum glycoproteins consisting of different monosaccharides. One of these is levo-fucose (L-fucose), a methyl pentose. Elevated levels of protein-bound fucose have been reported in various malignancies. Aim: The present study attempted to correlate levels of serum L-fucose as a biomarker with the various tumor node metastasis (TNM) stages of oral cancer. Methodology: The study was carried out on 90 subjects consisting of 30 healthy controls and 60 histopathologically proven oral squamous cell carcinoma (OSCC) cases. The serum fucose level estimation was done based on the method adopted by Winzler. Statistical analysis included independent sample's t-test, one-way ANOVA test, Karl–Pearson correlation test, and Tukey's HSD post hoc test to evaluate the significance and variability of values between groups. Results: Significant elevation in serum fucose levels was noticed among OSCC patients when compared with the controls and a progressive ascent of L-fucose levels were noted as the stage of severity increased. Serum fucose levels were independent of histopathological grading, age, and sex. Conclusion: Serum L-fucose levels were increased in OSCC patients, and a positive correlation was observed between serum L-fucose levels and TNM staging of OSCC. Thus, serum L-fucose can be used as an effective diagnostic and prognostic biomarker in OSCC patients.

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 (K_M 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.

α-L-Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α-L-fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli. Afc1 had the α-L-fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined. The putative acid/base residue of Afc1, Ser205, was replaced by a glutamic acid which is conserved in GH29-B α-L-fucosidases. However, the mutant Afc1-S205E still failed to show enzyme activity. Afc2 and Afc3 were determined to be 1,3-1,4-α-L-fucosidase of GH29-B subfamily and 1,2-α-L-fucosidase of GH95 family, respectively, and both of them could release fucose from porcine gastric mucin (PGM). When C. perfringens ATCC 13124 grew with the presence of PGM, the transcription of afc1 decreased slightly, while those of afc2 and afc3 increased to 2.2-fold and 1.4-fold, respectively, and the enzyme activities of Afc2 and Afc3 in the culture increased to 2.2-fold and 2.6-fold, respectively. These results suggest that Afc2 and Afc3 are involved in the degradation of intestinal fucosyl glycans by C. perfringens ATCC 13124.