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PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR LIPASES

TEHREEMA IFTIKHAR^{2, 1*}, MUBASHIR NIAZ², RUKHSANA JABEEN³AND IKRAM UL HAQ¹  

Abstract: The present study describes the enzyme produced after optimization of the cultural conditions subjected to ammonium sulfate precipitation for salting out the proteins. 60% ammonium sulfate showed the enzyme activity of 11.45 U mL^-1 by wild and 28.2 U mL^-1 by mutant strain of R. oligosporus var. microsporus while in 80% ammonium sulfate the enzyme activity by both the wild (13.14 U mL^-1) and mutant (29.5 U mL^-1) strains increased which indicates the partial purification of enzyme. Desalted enzyme was subjected to DEAE-cellulose column for ion exchange chromatography. Wild strain showed 206.73 fold purification and 82.56% recovery while the mutant strain showed 407.34 fold purification with 62.83% recovery. Sephadex G-100, is a cross-linked polymer used for gel filtration chromatography, which is used for differentiating the molecular size. There is 446.19 fold (W) and 710.02 fold (M) purification of enzyme which showed that most of the contamination proteins are removed. The effect of pH, temperature and metal ions was also investigated on the activity of purified lipases. It is evident from the results that the maximum residual activity by both strains 81% (W) and 100% (M) was observed in the reaction mixture of pH 8.0. The results showed that lipases retained 80% of its activity at 25^oC-30^oC by wild and 100% of its activity at 20^oC-50^oC by mutant strain of R. oligosporus var. microsporus. Mn⁺⁺ stimulated the activity of lipases by the wild while it has inhibitory effect on lipases activity of mutant strain. Other ions Like Ca⁺⁺, K⁺, Mg⁺⁺, Cu⁺⁺ and Na⁺ stimulated the activity of lipases by both wild and mutant strains. Both wild and mutant strains showed the same response of inhibition of enzyme activity in the presence of Hg⁺⁺ and Fe⁺⁺.