This study examined the recovery and preliminary biochemical characterization of extracellular lipase and catechol-cleaving dioxygenase activities from a bacterial isolate identified phenotypically as Pseudomonas luteola and obtained from petroleum-contaminated soil. Crude enzyme preparations were subjected to ammonium sulfate precipitation followed by gel filtration chromatography. For the dioxygenase preparation, specific activity increased from 15.3 U/mg in the crude extract to 588 U/mg after gel filtration, corresponding to a 38.4-fold enrichment with a final apparent yield of 15.9%. For lipase, specific activity increased from 0.00537 U/mg to 6.99 U/mg, corresponding to a 1302-fold enrichment, while the calculated apparent yield exceeded 100%, indicating increased measurable activity after purification and therefore requiring cautious interpretation. Chromatographic elution profiles showed discrete protein peaks associated with the pooled active fractions. The purified lipase displayed highest activity and stability at 30–35∘C and near-neutral pH, whereas the dioxygenase preparation showed maximum activity under mesophilic temperatures and around pH 7.0. Taken together, the results indicate that petroleum-associated bacterial isolates can provide enzyme preparations that remain active under mild conditions relevant to environmental biotechnology. The present work should be viewed as a preliminary purification and characterization study; it establishes a biochemical basis for future investigations of enzyme identity, catalytic performance, and direct hydrocarbon biodegradation.
This study examined the recovery and preliminary biochemical characterization of extracellular lipase and catechol-cleaving dioxygenase activities from a bacterial isolate identified phenotypically as Pseudomonas luteola and obtained from petroleum-contaminated soil. Crude enzyme preparations were subjected to ammonium sulfate precipitation followed by gel filtration chromatography. For the dioxygenase preparation, specific activity increased from 15.3 U/mg in the crude extract to 588 U/mg after gel filtration, corresponding to a 38.4-fold enrichment with a final apparent yield of 15.9%. For lipase, specific activity increased from 0.00537 U/mg to 6.99 U/mg, corresponding to a 1302-fold enrichment, while the calculated apparent yield exceeded 100%, indicating increased measurable activity after purification and therefore requiring cautious interpretation. Chromatographic elution profiles showed discrete protein peaks associated with the pooled active fractions. The purified lipase displayed highest activity and stability at 30–35∘C and near-neutral pH, whereas the dioxygenase preparation showed maximum activity under mesophilic temperatures and around pH 7.0. Taken together, the results indicate that petroleum-associated bacterial isolates can provide enzyme preparations that remain active under mild conditions relevant to environmental biotechnology. The present work should be viewed as a preliminary purification and characterization study; it establishes a biochemical basis for future investigations of enzyme identity, catalytic performance, and direct hydrocarbon biodegradation.
