Thesis (Ph.D.)--Chulalongkorn University, 2011

Synthetic dyes are extensively used in many industries. The effluents from these factories represent a major source of water pollution. Actually, physical and chemical methods are used for the treatment of these effluents but they are expensive and produce hazardous by- products. First, the decolorization of seven dyes with diverse chemical structure (azo, anthraquinone and indigo) was studied. An enzyme extract enriched in laccase from the fungus Trametes versicolor strain DSM11269 was found to decolorized the anthraquinone derivative dyes, Alizarin Red S and Remazol Brilliant Blue R, in three hours by 55% and 70%, respectively. The azo compounds (Amaranth, Cibacron Brilliant Red 3B-A, Direct Blue 71 and Reactive Black 5), and the indigo molecule (Indigo Carmine), showed a higher resistance to decolorization (<10% in 3 hours). Biotechnological and environmental applications require large amounts of enzymes and the secretion of laccase by fungal organism may not be suitable due to undesirable preparation procedure (such as presence of toxic inducers) and time consuming. So, heterologous expression of laccase in optimized hosts is a considerable way to produce the recombinant enzyme. The yeast Yarrowia lipolytica has been choosen as an efficient system for recombinant protein expression with high transformation efficiency. A cDNA encoding laccase (lcc1) was isolated from the white-rot fungus T. versicolor by RT-PCR. The cDNA encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 was expressed in Y. lipolytica under the control of the pTEF constitutive promoter. After purification by affinity chromatography, the recombinant laccase has an apparent molecular weight of ~75 kDa, comprised of ~20% N-linked glycans. The activity produced for the mutant L185P/Q214K (rM-4A) generated by molecular evolution was by six-fold higher than that obtained with the wild type enzyme. After purification, rM-4A mutant showed a 2.1-fold higher specific activity and a 1.7- and 2.4- fold enhancement of the kcat and kcat /Km values, respectively, with ABTS substrate and a 2.5- and 2.8-fold increase, respectively, with 2, 6-dimethoxyphenol substrate, compared to the wild type (rWT) laccase. Both the rWT and the rM-4A Lcc1 enzymes were stable over a broad pH range (3.6-7.6) and active at normal temperatures (>88% activity at 25-35oC after one hour). The rWT and mutant laccase preparation decolorize 95 and 97% of 74.6 µM Amaranth solution. The results revealed that decolorization rate is about 70 nM.s-1 in the presence of acetosyringone redox mediator.