Polyacrylamide gel electrophoresis of exoenzymes produced by Pseudomonas fluorescens strain W

Abstract

Culture filtrates from strain W of Pseudomonas fluorescens that cause lysis of gram-negative bacterial cell envelopes were examined for specific hydrolases. The enzymes were concentrated by ammonium sulfate fractionation and by column fractionation on Sephadex G-100. Attempts to separate the individual hydrolases quantitatively by elution from DEAE-cellulose failed because of the formation of aggregates. Resolution of the individual hydrolases was accomplished by disc gel electrophoresis using polyacrylamide (7.5%) gels buffered at pH 8.9 with Tris-glycine. The enzyme mixture was separated into 13 distinct protein bands which were stained with Coomassie blue. The individual hydrolases were detected either directly on the gels or by assay after elution from gel segments and included four proteinases, three phosphatases, two β-glucosidases, one ribonuclease, one lipase, one esterase, and one catalase. These methods provide a rapid, sensitive technique for the detection of many individual hydrolases in a complex mixture.