Alcalde Miguel, Bulter Thomas, Arnold Frances H
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA, USA.
J Biomol Screen. 2002 Dec;7(6):547-53. doi: 10.1177/1087057102238629.
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.
多环芳烃（PAHs）是广泛分布于陆地和水生环境中的高毒性有机污染物。在本研究中，基于对云芝和嗜热毁丝霉的真菌漆酶对12种芳烃的氧化作用的研究，开发了2种用于漆酶催化降解PAHs的比色测定法。作者使用硼氢化钠水溶液，可将漆酶催化蒽生物氧化的单一产物还原为橙色的9,10-蒽二氢醌，可通过分光光度法定量。还介绍了一种使用聚合染料（Poly R-478）作为在介质存在下漆酶降解木质素的替代底物的测定法。Poly R-478的脱色与漆酶介导的PAHs氧化相关。这表明木质素分解指示剂如Poly R-478可用于筛选降解PAHs的漆酶；它在定向进化实验中筛选突变体文库时也将很有用。Poly R-478稳定且易溶解。它具有高消光系数，对白腐真菌、酵母和细菌的毒性低，这使其可应用于固相测定形式。
