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.
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