Isolation and characterization of an alginate lyase from Klebsiella aerogenes.
The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37 degrees C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acid-rich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.
Lange
B
B
Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität, Bochum, Federal Republic of Germany.
Wingender
J
J
Winkler
U K
UK
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000254:growth & development; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D007202:Indicators and Reagents; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase