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Journal of Biomolecular Screening
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Colorimetric Assays for Biodegradation of Polycyclic Aromatic Hydrocarbons by Fungal Laccases

Miguel Alcalde

Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA.

Thomas Bulter

Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA.

Frances H. Arnold

Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants widely distributed in terrestrial and aquatic environments. In the present work, 2 colorimetric assays for laccase-catalyzed degradation of PAHs were developed based on studies of the oxidation of 12 aromatic hydrocarbons by fungal laccases from Trametes versicolor and Myceliophthora thermophila. Using a sodium borohydride water-soluble solution, the authors could reduce the single product of laccase-catalyzed anthracene biooxidation into the orange-colored 9,10-anthrahydroquinone, which is quantifiable spectrophotometrically. An assay using polymeric dye (Poly R-478) as a surrogate substrate for lignin degradation by laccase in the presence of mediator is also presented. The decolorization of Poly R-478 was correlated to the oxidation of PAHs mediated by laccases. This demonstrates that a ligninolytic indicator such as Poly R-478 can be used to screen for PAH-degrading laccases; it will also be useful in screening mutant libraries in directed evolution experiments. Poly R-478 is stable and readily soluble. It has a high extinction coefficient and low toxicity toward white rot fungi, yeast, and bacteria, which allow its application in a solid-phase assay format.

Journal of Biomolecular Screening, Vol. 7, No. 6, 547-553 (2002)
DOI: 10.1177/1087057102238629
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M. Alcalde, T. Bulter, M. Zumarraga, H. Garcia-Arellano, M. Mencia, F. J. Plou, and A. Ballesteros
Screening Mutant Libraries of Fungal Laccases in the Presence of Organic Solvents
J Biomol Screen, September 1, 2005; 10(6): 624 - 631.
[Abstract] [PDF]