Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Alginate lyases are enzymes that degrade alginate through beta-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 degrees 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 mu M, 22 +/- 5 mu M, 60 +/- 2 mu M, and 123 +/- 6 mu M, respectively. AlyA and AlyB were found principally to cleave the beta-1,4 bonds between beta-D-mannuronate and alpha-L-guluronate and subunits; AlyD and AlyE were found to principally cleave the alpha-1,4 bonds involving alpha-L-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.