3 Units at 37oC
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|Content:||3 Units at 37oC|
|Formulation:||In solution (Tris.HCl/NaCl/EDTA)|
|Stability:||> 3 years at 4oC|
|Synonyms:||1,2-alpha-L-fucosidase; 2-alpha-L-fucopyranosyl-beta-D-galactoside fucohydrolase|
|Concentration:||Supplied at ~ 12 U/mL|
|Expression:||Recombinant from a Microbial source|
|Specificity:||Highly specific for non-reducing terminal L-fucose residues linked to D-galactose residues by a 1,2-α-linkage. Does not hydrolyse p-nitrophenyl-α-L-fucopyranoside.|
|Specific Activity:||~ 70 U/mg (37oC, pH 6.5 on 2’-fucosyllactose)|
|Unit Definition:||One Unit of α-1,2-L-fucosidase activity is defined as the amount of enzyme required to release one µmole of α-L-fucose per minute from 2’-fucosyllactose (2 mM) in sodium phosphate buffer (100 mM), pH 6.5 at 37oC.|
|Application examples:||For use in glycobiology research.|
High purity recombinant 1,2-α-L-Fucosidase (microbial) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Enam, F. & Mansell, T. J. (2018). Cell chemical Biology, In Press.
Human milk oligosaccharides (HMOs) are important prebiotic complex carbohydrates with demonstrated beneficial effects on the microbiota of neonates. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of monosaccharide and linkage analysis. To enable high-throughput screening of HMO structures, we constructed a whole-cell biosensor that uses heterologous expression of glycosidases to generate linkage-specific, quantitative fluorescent readout for a range of HMOs at detection limits down to 20 µM in approximately 6 hr. We also demonstrate the use of this system for orthogonal control of growth rate or protein expression of particular strains in mixed populations. This work enables rapid non-chromatographic linkage analysis and lays the groundwork for the application of directed evolution to biosynthesis of complex carbohydrates as well as the prebiotic manipulation of population dynamics in natural and engineered microbial communities.Hide Abstract
Hykollari, A., Paschinger, K., Eckmair, B. & Wilson, I. B. (2017). High-Throughput Glycomics and Glycoproteomics: Methods and Protocols, 1503, 167-184.
N-glycans from invertebrates and protists have often unusual structures which present analytical challenges. Both core and antennal modifications can be quite different from the more familiar vertebrate glycan motifs; thereby, contrary to the concept that “simple” organisms have “simple” N-glycans, rather complex oligosaccharides structures, including zwitterionic and anionic ones, have been found in a range of species. Thus, to facilitate the optimized elucidation of the maximal possible range of structures, the analytical workflow for glycomics of these organisms should include sequential release and fractionation steps. Peptide:N-glycosidase F is sufficient to isolate N-glycans from fungi and some protists, but in most invertebrates core α1,3-fucose is present, so release of the glycans from glycopeptides with peptide:N-glycosidases A is required. Subsequent solid-phase extraction with graphitized carbon and reversed phase resins enables different classes of N-glycans to be separated prior to high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Depending on the types and numbers of glycans present, either reversed- or normal-phase HPLC (or both in series) enable even single isomeric or isobaric structures to be separated prior to MALDI-TOF MS and MS/MS. The use of enzymatic or chemical treatments allows further insights to be gained, although some glycan modifications (especially methylation) are resistant. Using a battery of methods, sometimes up to 100 structures from a single organism can be assigned, a complexity which raises evolutionary questions regarding the function of these glycans.Hide Abstract
Yan, S., Jin, C., Wilson, I. B. & Paschinger, K. (2015). Journal of Proteome Research, 14(12), 5291-5305.
Recent studies have shown a remarkable degree of plasticity in the N-glycome of the model nematode Caenorhabditis elegans; ablation of glycosylation-relevant genes can result in radically altered N-glycan profiles despite only minor biological phenotypic effects. Up to four fucose residues and five different linkages of fucose are known on the N-glycans of C. elegans. Due to the complexity in the wild type, we established three mutant strains defective in two core fucosyltransferases each (fut-1;fut-6, fut-1;fut-8, and fut-6;fut-8). Enzymatically released N-glycans were subject to HPLC and MALDI-TOF MS/MS, in combination with various treatments, to verify structural details. The N-glycome of the fut-1;fut-6 mutant was the most complex of the three double-mutant strains due to the extension of the core α1,6-fucose as well as the presence of fucose on the bisecting galactose. In contrast, maximally two fucoses were found on N-glycans of the fut-1;fut-8 and fut-6;fut-8 strains. The different locations and capping of fucose meant that up to 13 isomeric structures, many highly galactosylated, were determined for some single masses. These data not only show the high variability of the N-glycomic capacity of a “simple” nematode but also exemplify the need for multiple approaches to reveal individual glycan structures within complex invertebrate glycomes.Hide Abstract