ABSTRACT Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number ( k cat ) for alginate of 0.60 ± 0.02 s −1 , 3.7 ± 0.3 s −1 , 4.5 ± 0.5 s −1 , and 7.1 ± 0.2 s −1 , respectively. The K m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β- d -mannuronate and α- l -guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α- l -guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.