Characterization of the alginate lyases from Vibrio 1 splendidus 12 B 01 2 3

18 Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond 19 into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a 20 marine bacterioplankton species that can grow on alginate as its sole carbon source. We 21 identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases – 22 AlyA, AlyB, AlyD, and AlyE – from V. splendidus 12B01. The four lyases were found to have 23 optimal activity between pH 7.5-8.5 and at 20-25°C, consistent with their use in a marine 24 environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for 25 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 Km 26 values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, 27 and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds 28 between β-D-mannuronate and α-L-guluronate and subunits; AlyD and AlyE were found to 29 principally cleave the α-1,4 bonds involving α-L-guluronate subunits. The four alginate lyases 30 degrade alginate into longer chains of oligomers. 31