PARTIAL-PURIFICATION AND CHARACTERIZATION OF A MANNURONAN-SPECIFIC ALGINATE LYASE FROM PSEUDOMONAS-AERUGINOSA

Production of a thick exopolysaccharide coat (alginate) by mucoid stra ins of Pseudomonas aeruginosa has been shown to contribute to the path ogenicity and persistence of these bacteria in the lungs of patients w ith cystic fibrosis. Previous studies have shown that some mucoid P. a eruginosa strains produce an enzyme(s) capable of degrading this algin ate coat. In this study, an alginate lyase from mucoid P. aeruginosa s train WcM#2 was isolated and characterized. Lyase production was enhan ced by the addition of 0.2-0.3 M NaCl to the growth media. The lyase w as eluted from an alginate-Sepharose affinity column with 0.5 M NaCl, which can serve as a simple one-step purification protocol for obtaini ng semi-pure functional alginate lyase. Fractionation of the enzyme pr eparation on a Sephadex G-75 sizing column showed that the enzyme has an apparent molecular weight of 40,000, whereas sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested a molecular w eight of approximately 43,000. The affinity-purified enzyme had a pH o ptimum of 9.0, its activity was enhanced in the presence of 0.3 M NaCl , and it showed substrate specificity for polymannuronic acid blocks. These results demonstrate the presence of a mannuronan-specific algina te lyase in P. aeruginosa that differs in several respects from previo us reports of P. aeruginosa alginate lyases.