Lipase producing Aspergillus was isolated from soil collected from a refuse dump site located at Awka, Anambra State, Nigeria using standard microbiology and biochemical techniques. Crude extract of lipase was produce...Lipase producing Aspergillus was isolated from soil collected from a refuse dump site located at Awka, Anambra State, Nigeria using standard microbiology and biochemical techniques. Crude extract of lipase was produced after a successful screening of the isolates using mineral broth containing p-NPP through submerged fermentation system with optimized physiologic conditions. Three steps of purification were carried out: Ammonium sulphate, dialysis and gel filtration (sephadex G-150). Crude extract was precipitated by using 70% saturation of ammonium sulphate at pH 6.0 which gave the optimum precipitation of the protein with specific activity of 260.56 U/mg. Precipitation using ammonium sulphate carried out at pH 6.5 and 8.0 gave specific activity of 217 U/mg of the protein. The precipitates were further desalted through dialysis for twelve hours and specific activity of 343.20 U/mg was recorded from the dialysate afterwards. Further purification was done by using sephadex G-150 and specific activity of 490.55 U/mg was recorded from the active pooled fractions. The purification table showed a 2.32 purification folds of lipase was gotten after gel filtration (sephadex G-150) with a lipase percentage yield of 2.00%. The specific activity of lipase increased from 211.81 to 490.55 U/mg. Characterization of β-galactosidase gave optima pH and temperature of the enzyme at 6.0 and 60°C respectively. Kinetic constants: K<sub>m</sub> and V<sub>max</sub> values were obtained at various concentrations of p-NPP where 0.32 mM and V<sub>max</sub> of 200.00 μmol/min respectively. Ca<sup>2+</sup> and Co<sup>2+</sup> showed greater effect on lipase activity in a concentration-dependent manner (0.03 - 0.05 M) when compared to Mn<sup>2+</sup> and Fe<sup>2+</sup>. The results from this study have shown that lipase produced from filamentous Aspergillus has a wide range of activity over physiologic conditions in regards to industrial and clinical standard operational procedures.展开更多
文摘Lipase producing Aspergillus was isolated from soil collected from a refuse dump site located at Awka, Anambra State, Nigeria using standard microbiology and biochemical techniques. Crude extract of lipase was produced after a successful screening of the isolates using mineral broth containing p-NPP through submerged fermentation system with optimized physiologic conditions. Three steps of purification were carried out: Ammonium sulphate, dialysis and gel filtration (sephadex G-150). Crude extract was precipitated by using 70% saturation of ammonium sulphate at pH 6.0 which gave the optimum precipitation of the protein with specific activity of 260.56 U/mg. Precipitation using ammonium sulphate carried out at pH 6.5 and 8.0 gave specific activity of 217 U/mg of the protein. The precipitates were further desalted through dialysis for twelve hours and specific activity of 343.20 U/mg was recorded from the dialysate afterwards. Further purification was done by using sephadex G-150 and specific activity of 490.55 U/mg was recorded from the active pooled fractions. The purification table showed a 2.32 purification folds of lipase was gotten after gel filtration (sephadex G-150) with a lipase percentage yield of 2.00%. The specific activity of lipase increased from 211.81 to 490.55 U/mg. Characterization of β-galactosidase gave optima pH and temperature of the enzyme at 6.0 and 60°C respectively. Kinetic constants: K<sub>m</sub> and V<sub>max</sub> values were obtained at various concentrations of p-NPP where 0.32 mM and V<sub>max</sub> of 200.00 μmol/min respectively. Ca<sup>2+</sup> and Co<sup>2+</sup> showed greater effect on lipase activity in a concentration-dependent manner (0.03 - 0.05 M) when compared to Mn<sup>2+</sup> and Fe<sup>2+</sup>. The results from this study have shown that lipase produced from filamentous Aspergillus has a wide range of activity over physiologic conditions in regards to industrial and clinical standard operational procedures.
