Abstract:
This study was carried out with aim of ascertaining the substrate specificity of laccase from Pleurotus pulmonarius towards aromatic compounds. A pure culture of Pleurotus pulmonarius was obtained after it has been sub-cultured from its stock culture and grown on potatoes dextrose agar (PDA). The pure culture of Pleurotus pulmonarius showed a positive result when screened for laccases expression using 2, 2- C 3- ethyl-benzothazoline 6-sulfonate (ABTS) as substrate. A pilot study for laccase production was carried out for 14 days of which day nine (9) showed the day of maximum laccase activity of 590 U/ml. The total volume of 1000 ml of the enzyme was produced, in solid state fermentation system (SSFs) using wheat bran as the lignocellulosic based substrate, which served as the crude. The 80 % ammonium sulphate saturation was found suitable to precipitate protein with highest laccase specific activity of 3252 U/mg. After dialysis, the specific activity was observed to be 7310 U/mg. The purification folds after ammonium sulphate saturation, dialysis and gel filtration were 1.27, 2.85 and 3.71, respectively. The percentage yields after ammonium sulphate saturation, dialysis and gel filtration were 3.78, 2.68 and 1.90, respectively. Laccases from Pleurotus pulmonarius showed activity within the broad range of pH of (3.0 to 6.5) and temperature range of 30 ºC - 70 ºC. The optimum pH was 4.0 with an activity of 566.124 U/ml, while optimum temperature was observed at 50 0C with an activity of 557.234 U/ml. In the effect of substrate concentration, 90 µM of ABTS gave highest activity of 61.24 µmol/min, with Vmax of 90.91 µmol/min and KM of 31.27 µM. The laccase specificity for ABTS, α-Naphthol, 2, 6-DMP and O-dianisidine were found to be 2.91, 3.50, 20.83 and 27.03, respectively. The high specificity of laccase for various aromatic compounds makes laccase a promising enzyme for biodegradation of dyes which is eco-friendly.
