title: Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.
abstract: An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).
authors.affiliation: Marine Microbiology Department, National Institute of Oceanography and Fisheries (NIOF) , Alexandria , Egypt.
authors.forename: Ehab A
authors.initials: EA
authors.lastname: Beltagy
authors.affiliation: Botany and Microbiology Department, Faculty of Science, Alexandria University , Alexandria , Egypt.
authors.forename: Aliaa
authors.initials: A
authors.lastname: El-Borai
authors.affiliation: Botany and Microbiology Department, Faculty of Science, Alexandria University , Alexandria , Egypt.
authors.forename: Marina
authors.initials: M
authors.lastname: Lewiz
authors.affiliation: Botany and Microbiology Department, Faculty of Science, Alexandria University , Alexandria , Egypt.
authors.forename: Samy A
authors.initials: SA
authors.lastname: ElAssar
mesh_terms: D000464:Alginates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003636:DEAE-Cellulose / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation; D044223:Pseudomonas stutzeri / Q000201:enzymology*; D013696:Temperature; D014871:Water Microbiology
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D003911:Dextrans; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D003636:DEAE-Cellulose; C025614:sephadex; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
keywords: Alginate lyase; Enzyme properties; Enzyme purification; Pseudomonas sp.