44D-6 |
Isolation, purification and characterization of the laccase of Pleurotus ostreatus |
M. E. JARAMILLO-FLORES1, J. Aquino-Morales, and H. Hernández-Sánchez. (1) Dept. de Graduados e Investigación en Alimentos, Escuela Nacional de Ciencias Biológicas - IPN, Carpio y Plan de Ayala, Mexico, 11340, Mexico Pleurotus ostreatus contains many enzymes of the monophenol monooxigenase type catalyzing redox reactions with specific substrates. The PPO discovered by Schoenbein in 1856 in mushrooms are proteins with copper in the active site catalyzing the o-hydroxilation of monophenols to o-dihydroxiphenols in the presence of molecular oxygen and a hydrogen donor and the oxidation of odihydrophenols to o-benzoquinones also in the presence of molecular oxygen. The presence of these enzymes in Pleurotus ostreatus represents a problem for its commercialization since a dark product is not easily accepted. The objective of this research was to isolate and partially purify the monophenol monooxigenase (laccase) of P. ostreatus. The spawn was produced in wheat grains using a pure culture of the fungus as inoculum. The fruiting bodies were obtained in straw. The extraction, isolation and partial purification of the enzyme were done by precipitation with ammonium sulfate followed by column chromatography (Sephadex G-150) and by ion exchange chromatography (Econo-Pac High Q). Enzyme kinetics studies were performed with the purified laccase. The relative molecular weight was 76 kDa, and the values of the kinetic constants were: Km=32.857 mM and Vmax=43.29 AU using catechol as a substrate and Km=47.959 mM and Vmax=36.9 AU when the 3,4-dihydroxibenzoic acid was used as a substrate. This work can be useful in future investigations focused on laccase crystallization and 3-D structure determination by NMR or X-ray crystallography.
Session 44D, International
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