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|$\text{L} $| -Fucose is the most widely distributed |$\text{L} $| -hexose in marine and terrestrial environments and presents a variety of functional roles. |$\text{L} $| -Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α- |$\text{L} $| -fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α- |$\text{L} $| -fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2′- and 3-fucosyllactose, while Fp284 degraded 2′-fucosyllactose and Fuc(α 1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α 1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6–6.0 and 25°C, hydrolyzing Fuc(α 1,4)GlcNAc with k cat = 1.3 s−1 and K m = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored. [ABSTRACT FROM AUTHOR]

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α‐1,4‐fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. Database: The assembled metagenome and raw sequence data is available at EMBL‐EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451–MH755453. Enzymes belonging to the GH107 family degrade sulfated fucans—complex polysaccharides found in brown macroalgae. However, currently very few members have been identified. This study describes the discovery of three new GH107 fucanases, verified by sequence and functional analyses. The work will aid in further understanding the structure of brown algal polysaccharides and the hydrolytic mechanisms involved in their degradation. [ABSTRACT FROM AUTHOR]

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both b-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems. [ABSTRACT FROM AUTHOR]