Ulvan is a complex water-soluble sulfated polysaccharide in the cell wall of green algae belonging to genus Ulva. It is composed of L-rhamnose-3-sulfate (Rha3S), glucuronic acid (GluA), iduronic acid (IduA), and D-xylose (Xyl) distributed in three repetition moieties. The first step of a bacterial ulvan degradation is the cleavage of the beta-glycosidic bond between Rha3S and GluA/IduA through a beta-elimination mechanism by a ulvan lyase to produce oligo-ulvans with unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronate (Delta) at the non-reducing end. We have identified an ulvan associated polysaccharide utilization locus (PUL) residing between two ulvan lyase genes belonging to families of polysaccharide lyase 24 (PL24) and PL25 in the genome of a ulvan-utilizing bacterium Glaciecola KUL10 strain. The PUL contains many genes responsible for oligo-ulvan degradation. Among them, we demonstrated that both KUL10_26540 and KUL10_26770 had an unsaturated beta-glucuronyl hydrolase activity to produce Rha3S and oligosaccharides, such as Rha3S-GluA-Rha3S, Rha3S-IduA-Rha3S and, Rha3S-Xyl-Rha3S, by releasing 5-dehydro-4-deoxy-D-glucuronate. KUL10_26540 showed much higher activity than KUL10_26770 and was more active on disaccharide than tetrasaccharide. We also found a rhamnosidase activity on four KUL10 gene products, although they could not react on the sulfated rhamnose. (C) 2019 Elsevier Inc. All rights reserved.