title: Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations.
abstract: Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) from Vibrio splendidus 12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-β-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showed kcat/Km from 2,130 mg(-1) ml s(-1) for the trisaccharide to 224 mg(-1) ml s(-1) for larger oligomers of ∼50 residues, and 50.5 mg(-1) ml s(-1) for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul, Republic of Korea Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Sujit Sadashiv
authors.initials: SS
authors.lastname: Jagtap
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Hehemann
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Polz
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul, Republic of Korea.
authors.forename: Jung-Kul
authors.initials: JK
authors.lastname: Lee
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA zhao5@illinois.edu.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D000464:Alginates / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D003470:Culture Media; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase