α-L-Fucosidases and their applications for the production of fucosylated human milk oligosaccharides.

α-L-Fucosidases (EC 3.2.1.51), catalyzing the hydrolysis of fucosides and/or the transfer of fucosyl residue, have been characterized and modified into a trans-fucosylation mode or, further, engineered to function as "fucosynthase", which can be employed for the enzymatic synthesis of bioactive glycans, including fucosylated human milk oligosaccharides (HMOs). More than half of HMOs are fucosylated and have attracted ever-increasing interest because of their excellent physiological functions on breast-fed infants. To date, the characterization of novel fucosidases and molecular modification of these enzymes have been extensively studied to efficiently synthesize valuable fucosylated compounds. Herein, we discuss the advantages and challenges of different strategies for the production of HMOs and compare various donor/acceptor substrates used for the synthesis of fucosylated HMOs and their biomimetics. The implementation of trans-fucosylation patterns investigated in this paper via well-designed fucosidase mutants and proper reaction conditions may lead to development of an excellent platform, serving both fundamental studies and industrial-scale processes, for valuable carbohydrates synthesis.Key Points• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.• Highlights different approaches for the production of human milk oligosaccharides.• Summarizes α-l-fucosidases and their mutants in enzymatic synthesis of fucosylated human milk oligosaccharides and the biomimetics.• Concludes future perspectives on methods for improving fucosylated compounds synthesis.

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides.

The α-1,3/4-l-fucosidases (EC 3.2.1.111; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of BbAfcB with the corresponding 17-aminoacid α-helical loop of CpAfc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT BbAfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4-l-fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ±â€¯2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT BbAfcB (14 ±â€¯0.3%) and comparable to that obtained with CpAfc2 (50 ±â€¯8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto-N-neotetraose (LNnT) could be enriched with LNFP III.

Proteomic analysis of scallop hepatopancreatic extract provides insights into marine polysaccharide digestion.

Marine polysaccharides are used in a variety of applications, and the enzymes that degrade these polysaccharides are of increasing interest. The main food source of herbivorous marine mollusks is seaweed, and several polysaccharide-degrading enzymes have been extracted from mollusk digestive glands (hepatopancreases). Here, we used a comprehensive proteomic approach to examine the hepatopancreatic proteins of the Zhikong scallop (Chlamys farreri). We identified 435 proteins, the majority of which were lysosomal enzymes and carbohydrate and protein metabolism enzymes. However, several new enzymes related to polysaccharide metabolism were also identified. Phylogenetic and structural analyses of these enzymes suggest that these polysaccharide-degrading enzymes may have a variety of potential substrate specificities. Taken together, our study characterizes several novel polysaccharide-degrading enzymes in the scallop hepatopancreas and provides an enhanced view of these enzymes and a greater understanding of marine polysaccharide digestion.

Fucosyllactose and L-fucose utilization of infant Bifidobacterium longum and Bifidobacterium kashiwanohense.

BACKGROUND: Human milk oligosaccharides (HMOs) are one of the major glycan source of the infant gut microbiota. The two species that predominate the infant bifidobacteria community, Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, possess an arsenal of enzymes including α-fucosidases, sialidases, and ß-galactosidases to metabolise HMOs. Recently bifidobacteria were obtained from the stool of six month old Kenyan infants including species such as Bifidobacterium kashiwanohense, and Bifidobacterium pseudolongum that are not frequently isolated from infant stool. The aim of this study was to characterize HMOs utilization by these isolates. Strains were grown in presence of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), and Lacto-N-neotetraose (LNnT). We further investigated metabolites formed during L-fucose and fucosyllactose utilization, and aimed to identify genes and pathways involved through genome comparison. RESULTS: Bifidobacterium longum subsp. infantis isolates, Bifidobacterium longum subsp. suis BSM11-5 and B. kashiwanohense strains grew in the presence of 2'-FL and 3'- FL. All B. longum isolates utilized the L-fucose moiety, while B. kashiwanohense accumulated L-fucose in the supernatant. 1,2-propanediol (1,2-PD) was the major metabolite from L-fucose fermentation, and was formed in equimolar amounts by B. longum isolates. Alpha-fucosidases were detected in all strains that degraded fucosyllactose. B. longum subsp. infantis TPY11-2 harboured four α-fucosidases with 95-99 % similarity to the type strain. B. kashiwanohense DSM 21854 and PV20-2 possessed three and one α-fucosidase, respectively. The two α-fucosidases of B. longum subsp. suis were 78-80 % similar to B. longum subsp. infantis and were highly similar to B. kashiwanohense α-fucosidases (95-99 %). The genomes of B. longum strains that were capable of utilizing L-fucose harboured two gene regions that encoded enzymes predicted to metabolize L-fucose to L-lactaldehyde, the precursor of 1,2-PD, via non-phosphorylated intermediates. CONCLUSION: Here we observed that the ability to utilize fucosyllactose is a trait of various bifidobacteria species. For the first time, strains of B. longum subsp. infantis and an isolate of B. longum subsp. suis were shown to use L-fucose to form 1,2-PD. As 1,2-PD is a precursor for intestinal propionate formation, bifidobacterial L-fucose utilization may impact intestinal short chain fatty acid balance. A L-fucose utilization pathway for bifidobacteria is suggested.

Comparative and phylogenetic analysis of alpha-L-fucosidase genes.

Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fucosidases that are present in all vertebrates likely reflect a distinct biological role for paralogous genes. Comparative sequence analysis of all metazoan alpha-L-fucosidases reveals a broad conservation of features, including the aspartate residue that constitutes the catalytic nucleophile. However, a cysteine which is thought to be part of the active site is also conserved in metazoa but not in arthropods, where it is replaced by an alanine. Phylogenetic analysis suggests a gene duplication event very early in metazoan evolution with the subsequent differential loss of isoforms in various metazoan lineages.

Progressive cone dystrophy associated with low alpha-L-fucosidase activity in serum and leukocytes.

The enzyme activities of acid phosphatase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and alpha-D-mannosidase were not significantly different in patients with myopia, retinal detachment, hereditary macular dystrophy, and unusual progressive cone dystrophy. alpha-L-Fucosidase activity in sera was lower in three patients with myopia and in two patients with unusual progressive cone dystrophy than in most of the others. Leukocytic alpha-L-fucosidase activity was lower in those with unusual progressive cone dystrophy. The two unrelated patients with unusual progressive cone dystrophy had slowly deteriorating visual acuity, color vision, and photopic electroretinographic responses, but ophthalmoscopically normal fundi and noncontributory family histories.