PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ORGANIC SOLVENT-TOLERANT LIPASE PRODUCED BY Pseudomonas fluorescens RB02-3 ISOLATED FROM MILK

Abstract

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50°C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd²⁺, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.

Keywords:

• enzyme characterization
• lipase
• milk
• organic solvent
• Pseudomonas fluorescens
• purification

ACKNOWLEDGMENTS

The work forms a part of the master of science thesis of Rukiye Boran.

Notes

Note. Lipase preparation was incubated in the presence of 25% (v/v) organic solvents for 1, 2, and 24 hr at 30°C on a rotary shaker. All assay were performed in duplicate. Relative activity was measured using a standard method with p-NPP and the activity of enzyme without added organic solvent was taken as 100%.

Note. Pseudomonas fluorescens RB02-3 lipase was incubated with various metal ions at 30°C for 30 min before the activity was measured with p-NPP. All assay were performed in duplicates. Relative activity of enzyme without any metal ion was defined as 100%.

^a Final concentration was 1 mM.

Note. Psuedomonas fluorescens RB02-3 lipase was incubated with various reagents at 30°C for 30 min before the activity was measured with p-NPP. All assay were performed in duplicates. Relative activity of enzyme without any reagent was defined as 100%.

^a Final concentration was 5 mM.