Discovery and characterization of novel fucoidanases for depolymerization of Macrocystis pyrifera fucoidan

Brown algae are central players in marine ecology and a rich, renewable resource with a millennia-long history of human use. Their extracellular matrix contains unique anionic polysaccharides, including fucoidans, recognized both as promising therapeutic agents and important players in biogeochemical carbon cycling. Fucoidans are structurally diverse and complex, and still not fully resolved at the molecular level, a challenge that has long hindered their systematic study and translation into biotechnological applications. This structural ambiguity has also contributed to inconsistent terminology within the field. Here, we outline the historical development of fucoidan structural characterization and nomenclature, identify recurring semantic inconsistencies, and propose a standardized terminology. Furthermore, we developed a mild extraction procedure that preserves native fucoidan structure and yields a high-molecular-weight, highly sulfated fucan from Macrocystis pyrifera biomass. Using this material as substrate for enzyme discovery enabled the identification of six novel fucoidanases, fucoidan-degrading enzymes, belonging to families GH107 and GH168. These enzymes originate from specialized fucoidan degrading genus Lentimonas spp. and a deep-sea hydrothermal vent metagenome. Comprehensive analytical characterization revealed distinct substrate specificities, reaction rates, and depolymerization mechanisms among these enzymes. The GH107 enzyme P5AFcnA exhibited the highest activity and was selected for detailed mechanistic and structural studies. Time-resolved analyses demonstrated that P5AFcnA depolymerizes M. pyrifera fucoidan through an endolytic mode of action, generating oligosaccharide intermediates that are further degraded into two sulfated tetramers bearing five and six sulfate esters, as well as a fucose-2,4-disulfate monomer. A recalcitrant fraction enriched in galactose remained undigested. Together, these findings highlight the importance of gentle extraction methods for preserving native fucoidan structure and thus enabling enzyme characterization. Beyond expanding the enzymatic toolbox for fucoidan processing, this work provides a comprehensive structural analysis of M. pyrifera fucoidan and advances conceptual clarity in the field through a proposed standardized nomenclature system. • A gentle extraction procedure for fucoidan was developed • The purified fucoidan had high molecular weight was highly sulfated • P5AFcnA hydrolyzed M. pyrifera fucoidan through an endolytic mechanism • End products were two tetramers and a fucose-2,4-disulfate monomer • Seven novel fucoidan-degrading enzymes were identified