Combined action of a bacterial monooxygenase and a fungal laccase for the biodegradation of mono- and poly-aromatic hydrocarbons.

The combined action of a wide substrate range toluene o-xylene monooxygenase from Pseudomonas sp. OX1, able to convert many aromatic compounds into mono- and di-hydroxylated derivatives, and fungal laccases from Pleurotus ostreatus which oxidize these hydroxylated products yielding polymers with reduced toxicity is described. This strategy permits to overcome many of the substrate specificity problems and dead end toxic products formation generally encountered in complex bacterial biodegradation pathways. Toluene and naphthalene degradations were tested as representative of mono- and poly-aromatic pollutants. The combined biological action was optimized in micellar and microemulsion systems able to increase the bioavailability of the hydrophobic aromatic pollutants. This approach allows efficient hydroxylations of hydrophobic substrates thus favoring the further action of fungal oxidases.

Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinus.

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, beta = 97.7 degrees , and contain 46% solvent. A complete native data set was collected to 1.4 A resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes.