Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium

120

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH 2 ). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH 2 extended the reactions catalyzed by these enzymes to the production of H 2 O 2 , the oxidation of Mn 2؉ , the reduction of Fe 3؉ , and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.Lignin is an aromatic heteropolymer of phenyl-propanoid units which confers structural rigidity to woody plant tissues and protects them from microbial attack (25). To depolymerize and mineralize lignin, white rot fungi have developed a nonspecific oxidative system including several extracellular oxidoreductases, low-molecular-weight metabolites, and activated oxygen species (47). The ability of white rot fungi to degrade a wide number of organopollutants is in part due to the action of this nonspecific system (42). Extracellular enzymes involved in the degradation of lignin and xenobiotics by white rot fungi include several kinds of laccases (34, 49), peroxidases (8, 33), and oxidases producing H 2 O 2 (20,32,50).The enzymatic composition of the ligninolytic system depends on the fungal species, with laccase being the common component (24,43). For this reason, a wide number of studies have focused on demonstrating the participation of laccase in significant ligninolytic events which were first attributed to other enzymes of the ligninolytic system. These events include the oxidation of nonphenolic lignin units, which comprise ca. 80% of the polymer, the generation of the H 2 O 2 required for both peroxidase activities and hydroxyl radical ( ⅐ OH) formation, and the production of Mn 3ϩ from the Mn 2ϩ pr...

Section: Discussionsupporting

confidence: 86%

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confidence: 99%

Induction, Isolation, and Characterization of Two Laccases from the White Rot BasidiomyceteCoriolopsis rigida

Saparrat

Guillén

Arambarri

et al. 2002

120

“…The ligninolytic enzymes, however, act as an indirect source of ⅐ OH through the generation of Fe 3ϩ and O 2 reductants, such as formate (CO 2 ⅐ Ϫ ) and semiquinone (Q ⅐ Ϫ ) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in ⅐ OH production, oxalate was used to generate CO 2 ⅐ Ϫ in a LiP reaction mediated by veratryl alcohol (4 (14). In this case, ⅐ OH radicals were generated by a semiquinone-driven Fenton reaction, as Q ⅐ Ϫ radicals were the main agents accomplishing Fe 3ϩ reduction.…”

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confidence: 99%

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

Gómez-Toribio

García-Martín

Martı́nez

et al. 2009

135

. In all cases, ⅐ OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H 2 O 2 levels and Fe 3؉ 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 ⅐ 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 ⅐ 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 ⅐ OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.

“…Except for O 2 ⅐Ϫ , all of these compounds are able to oxidize lignin units. However, the O 2 ⅐Ϫ produced by white-rot fungi (12) can participate in the production of H 2 O 2 via both dismutation ( (4). Furthermore, by reacting with phenoxyl radicals produced from lignin model compounds, it can result in oxidative degradation being favored over coupling reactions (15).…”

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confidence: 99%

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

Guillén

Muñoz

Gómez-Toribio

et al. 2000

105

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH 2 ) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH 2 ) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH 2 more efficiently than it oxidized MBQH 2 ; both the affinity and maximal velocity of oxidation were higher for DBQH 2 than for MBQH 2 . Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (