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Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production

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Abstract

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of “alpeorujo” (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

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Acknowledgements

This work was supported by the Spanish projects S-0505/AMB0100, A/019945/08, AGL2008-00572/AGR and P06-AGR-01906. R. Díaz thanks the Spanish Ministry for mobility fellowship to the Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.

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Authors and Affiliations

  1. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Prof. Albareda 1, 18008, Granada, Spain

    Rosario Díaz, Inmaculada García-Romera & Juan Antonio Ocampo

  2. Instituto de Fisiología Vegetal, Universidad Nacional de La Plata—Consejo Nacional de Investigaciones Científicas y Técnicas, La Plata, Argentina

    Mario C. N. Saparrat

  3. Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maeztu 9, 28040, Madrid, Spain

    Miguel Jurado & María Jesús Martínez

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  1. Rosario Díaz
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  2. Mario C. N. Saparrat
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  3. Miguel Jurado
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  4. Inmaculada García-Romera
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  5. Juan Antonio Ocampo
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  6. María Jesús Martínez
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Correspondence to María Jesús Martínez.

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Díaz, R., Saparrat, M.C.N., Jurado, M. et al. Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production. Appl Microbiol Biotechnol 88, 133–142 (2010). https://doi.org/10.1007/s00253-010-2723-z

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  • DOI: https://doi.org/10.1007/s00253-010-2723-z

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