Random Mutagenesis Methods for Hyper-Production of Laccase and its Effect on Azo Dye Decolourization

摘要

This work focuses on the improvement of the wild strain for the hyper production of laccase through random mutagenesis. Maximum laccase production (46956 U/L) was observed in mutant T. hirsuta, when rice bran (5 g) at 75 % moisture (w/v) was used with 1 mL inoculum at pH 6 and 25 °C in the presence of 1 % (w/v) glucose as a carbon source, 0.1 % (wAO sodium nitrate as a nitrogen source, 2 % of (v/v) glycerol as an inducer in submerged fermentation under static conditions. Laccase purification was performed by precipitating the enzyme with 90 % ammonium sulphate followed by ion exchange chromatography. The puried laccase had an apparent molecular mass of 49.9 kDa with monomelic structure estimated by SDS-PAGE. The three azo dyes could be efficiently decolourized by the purified laccase in the presence of a HBT as redox mediators after 3 h of incubation at pH 5. Laccase mediated degradation of poneaceau was monitored by UV-visible, IR-spectroscopy, HPLC and GCMS. Furthermore, the decolourization products of poneaceau were found to be aromatic amines and less phytotoxic nature than the untreated dye.