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Applied and Environmental Microbiology, June 2009, p. 3944-3953, Vol. 75, No. 12
0099-2240/09/$08.00+0 doi:10.1128/AEM.02137-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Ana B. García-Martín,2
María J. Martínez,1
Ángel T. Martínez,1 and
Francisco Guillén2*
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain,1 Departamento de Microbiología y Parasitología, Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain2
Received 15 September 2008/ Accepted 13 April 2009
A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe3+-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H2O2 + Fe2+
OH + OH– + Fe3+). H2O2 production, Fe3+ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H2O2 levels and Fe3+ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.
Published ahead of print on 17 April 2009.
Present address: Varian Iberica S.L., Avda. Pedro Diez 25, 28019 Madrid, Spain.
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