title: An oxidant and organic solvent tolerant alkaline lipase by P. aeruginosa mutant: downstream processing and biochemical characterization.
abstract: An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45 °C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca(2+), Mg(2+) and Na(+) tend to increase the enzyme activity, whereas, Fe(2+) and Mn(2+) ions resulted in discreet decrease in the activity. Divalent cations Ca(+2) and Mg(+2) seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca(+2) (5 mM) the optimum temperature shifted from 45 °C to 55 °C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of K(m) and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL(-1) and 5000 μg.mL(-1).min(-1), respectively.
authors.affiliation: Centre of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad, U.P., India.
authors.forename: Deepali
authors.initials: D
authors.lastname: Bisht
authors.affiliation: Centre of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad, U.P., India.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Yadav
authors.affiliation: Centre of Excellence, Department of Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad, U.P., India.
authors.forename: Nandan Singh
authors.initials: NS
authors.lastname: Darmwal
mesh_terms: D002412:Cations / Q000378:metabolism; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D020536:Enzyme Activators; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012997:Solvents / Q000378:metabolism; D013696:Temperature
chemical_list: D002412:Cations; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D016877:Oxidants; D012997:Solvents; D008049:Lipase
keywords: alkaline lipase; detergent stability; mutant; organic-solvent; p-nitrophenyl palmitate Pseudomonas