Induction, isolation, and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida.
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.
Saparrat
Mario C N
MC
Facultad de Ciencias Naturales y Museo, Instituto de Botánica Spegazzini, Universidad Nacional de La Plata, 53 477, 1900 La Plata, Argentina.
Guillén
Francisco
F
Arambarri
Angélica M
AM
Martínez
Angel T
AT
Martínez
María Jesús
MJ
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000378:metabolism; D052160:Benzothiazoles; D003300:Copper / Q000494:pharmacology; D003470:Culture Media; D004790:Enzyme Induction; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D042845:Laccase; D008031:Lignin / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism
D052160:Benzothiazoles; D003470:Culture Media; D006873:Hydroquinones; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D003300:Copper; D008031:Lignin; D010088:Oxidoreductases; D042845:Laccase