Functional exploration of the GH29 fucosidase family.

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family's large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Biochemical characterization of a novel α-L-fucosidase from Pedobacter sp. and its application in synthesis of 3'-fucosyllactose and 2'-fucosyllactose.

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2'-fucosyllactose (α-L-Fuc-(1,2)-D-Galß-1,4-Glu, 2'FL) and its isomer 3'-fucosyllactose (α-L-Fuc-(1,3)-D-Galß-1,4-Glu, 3'FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant α-L-fucosidase. An α-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported α-L-fucosidases. The purified α-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC), 3'FL (8.9 U/mg), and 2'FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 °C, respectively. PbFuc catalyzed the synthesis of 3'FL and 2'FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2'FL and 3'FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2'FL and 3'FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds.Key Points• A novel α-L-fucosidase (PbFuc) from Pedobacter sp. was cloned and expressed.• PbFuc showed the highest hydrolysis activity at pH 5.0 and 35 °C, respectively.• It was used for synthesis of 3'-fucosyllactose (3'FL) and 2'-fucosyllactose (2'FL).• The mixture of 3'FL and 2'FL promoted the growth of some Lactobacillus sp. and Bifidobacteria sp.

Synthesis of Fucosyl-Oligosaccharides Using α-l-Fucosidase from Lactobacillus rhamnosus GG.

Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of α-lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since α-lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using α-l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata.

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 Km value 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 kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of 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.

First characterization of fucosidases in spiders.

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

Purification, expression and characterization of a novel α-l-fucosidase from a marine bacteria Wenyingzhuangia fucanilytica.

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.

Rice α-fucosidase active against plant complex type N-glycans containing Lewis a epitope: purification and characterization.

Rice α-fucosidase (α-fucosidase Os, 58 kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plant N-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plant N-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-P-T-P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400).

Alpha-L-fucosidase isoenzyme iso2 from Paenibacillus thiaminolyticus.

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, constructed in E.coli DH5α cells, nucleotide sequence of a new 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-fucosidase iso2 were tested using different acceptor molecules. 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 important for different biochemical disciplines involving molecular modelling.

Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

α-Fucosidases with different substrate specificities from two species of Fusarium.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.

Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-ß-glycosidase and ß-fucosidase.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-ß-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a ß-glucosidase resulted in the unexpected generation of a thermostable, highly specific ß-fucosidase, concomitant with large increases in ß-glucosidase, ß-cellobiosidase, α-arabinofuranosidase, ß-mannosidase, ß-glucuronidase, ß-xylopyranosidase, and ß-fucosidase activities and a dramatic decline in 6-phospho-ß-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-ß-glycosidase and to generate a thermostable ß-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

Utilization of natural fucosylated oligosaccharides by three novel alpha-L-fucosidases from a probiotic Lactobacillus casei strain.

Three putative α-L-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2'-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3'-, 4'-, and 6'-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa.

Transglycosylating ß-d-galactosidase and α-l-fucosidase from Paenibacillus sp. 3179 from a hot spring in East Greenland.

Thermal springs are excellent locations for discovery of thermostable microorganisms and enzymes. In this study, we identify a novel thermotolerant bacterial strain related to Paenibacillus dendritiformis, denoted Paenibacillus sp. 3179, which was isolated from a thermal spring in East Greenland. A functional expression library of the strain was constructed, and the library screened for ß-d-galactosidase and α-l-fucosidase activities on chromogenic substrates. This identified two genes encoding a ß-d-galactosidase and an α-l-fucosidase, respectively. The enzymes were recombinantly expressed, purified, and characterized using oNPG (2-nitrophenyl-ß-d-galactopyranoside) and pNP-fucose (4-nitrophenyl-α-l-fucopyranoside), respectively. The enzymes were shown to have optimal activity at 50°C and pH 7-8, and they were able to hydrolyze as well as transglycosylate natural carbohydrates. The transglycosylation activities were investigated using TLC and HPLC, and the ß-d-galactosidase was shown to produce the galactooligosaccharides (GOS) 6'-O-galactosyllactose and 3'-O-galactosyllactose using lactose as substrate, whereas the α-l-fucosidase was able to transfer the fucose moiety from pNP-fuc to lactose, thereby forming 2'-O-fucosyllactose. Since enzymes that are able to transglycosylate carbohydrates at elevated temperature are desirable in many industrial processes, including food and dairy production, we foresee the potential use of enzymes from Paenibacillus sp. 3179 in the production of, for example, instant formula.

Identification of essential residues of human alpha-L-fucosidase and tests of its mechanism.

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of alpha-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

Two distinct alpha-L-fucosidases from Bifidobacterium bifidum are essential for the utilization of fucosylated milk oligosaccharides and glycoconjugates.

Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

Semen sialic acid surge and modulation of alpha-L-fucosidase activity: possible link to loss in reproductive capacity during trypanosomiasis.

The profiles of semen sialic acid and the enzyme alpha-L-fucosidase were studied in rams undergoing chronic infection by Trypanosoma congolense. Our data showed a significant surge in the level of sialic acid with parasitaemia. The pattern followed a polynomial function we had reported for erythrocyte sialic acid in mice undergoing acute infection by T. congolense. The activity of the enzyme alpha-fucosidase decreased progressively with approximately 60% decrease at the end of the 14 weeks of infection. Representative semen samples from the control and infected rams were subjected to kinetic characterization. While the uninfected semen sample showed two active pH peaks at 4.5-5.5 and at 6.8-7.2, respectively, there was an apparent shift to only a single pH optimum at 4.5-5.5 for the pathological semen. The fucosidases from both sources were optimally active at 35 degrees C albeit with contrasting activation energies (E(a)) with values 20.58 and 35 kJ/mol for the control and infected semen, respectively. Kinetic studies using methylumbelliferyl-beta-fucoside (4MU-Fuc) as substrate gave K(M) and V(max) values of 3.25 microM and 14.6 micromol. min(-1) mg(-1), respectively for the control semen. The values for the infected semen were 18.25 microM and 10.5 micromol. min(-1) mg(-1), respectively. The significance of these results is discussed as they relate to loss in reproductive capacity in trypanosomoses.

A Novel alpha1,2-L-fucosidase acting on xyloglucan oligosaccharides is associated with endo-beta-mannosidase.

Endo-beta-mannosidase, which hydrolyses the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, was discovered in plants. During the course of the purification of the enzyme from lily flowers, we found a higher molecular mass form of the enzyme (designated as EBM II). EBM II was purified by column chromatography to homogeneity and its molecular composition revealed EBM II to be comprised of endo-beta-mannosidase and an associated protein. The cDNA of this associated protein encodes a protein with slight homology to the fucosidase domain of bifidus AfcA. EBM II has alpha1,2-L-fucosidase activity and acts on a fucosylated xyloglucan nonasaccharide. The amino acid sequence of this associated protein has no similarity to known plant alpha-L-fucosidases. These results show that EBM II is a novel alpha1,2-L-fucosidase and a protein complex containing endo-beta-mannosidase.

Characterization of a new α-l-fucosidase isolated from Fusarium proliferatum LE1 that is regioselective to α-(1 → 4)-l-fucosidic linkage in the hydrolysis of α-l-fucobiosides.

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 (pNPFuc), with Km = 1.1 ± 0.1 mM and kcat = 39.8 ± 1.8 s-1. l-fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited pNPFuc 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 â†’ 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 pNP-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.

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I.

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.

Purification and characterization of alpha-L-fucosidase from human primary hepatocarcinoma tissue.

AIM: To purify and characterize alpha-L-fucosidase from human liver cancer tissue and to detect the localization of alpha-L-fucosidase in tumor tissue. METHODS: Cation exchange chromatography on CM-52 and ultrafiltration were used to separate alpha-L-fucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-alpha-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues. RESULTS: Human alpha-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue. CONCLUSION: The purified alpha-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties from alpha-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.