Recombinant Laccase: I. Enzyme cloning and characterization

Authors

  • Claudio Nicolini,

    Corresponding author
    1. Biophysics and Nanobiotechnology Laboratories, DIMES, University of Genoa, Genoa, Italy
    2. Nanoworld Institute Fondazione EL.B.A Nicolini, Pradalunga, Bergamo, Italy
    3. Biodesign Institute, Arizona State University, Tempe, Arizona
    • President Nanoworld Institute Fondazione ELBA Nicolini, Eminent Chair of Biophysics at University of Genova, Genova, Italy; Foreign Member Russian Academy of Sciences, Moscow, Russia.
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  • Debora Bruzzese,

    1. Nanoworld Institute Fondazione EL.B.A Nicolini, Pradalunga, Bergamo, Italy
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  • Maria Teresa Cambria,

    1. Nanoworld Institute Fondazione EL.B.A Nicolini, Pradalunga, Bergamo, Italy
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  • Nicola Luigi Bragazzi,

    1. Biophysics and Nanobiotechnology Laboratories, DIMES, University of Genoa, Genoa, Italy
    2. School of Public Health, Department of Health Sciences (DISSAL), University of Genoa, Genoa, Italy
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  • Eugenia Pechkova

    1. Biophysics and Nanobiotechnology Laboratories, DIMES, University of Genoa, Genoa, Italy
    2. Nanoworld Institute Fondazione EL.B.A Nicolini, Pradalunga, Bergamo, Italy
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Abstract

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. J. Cell. Biochem. 114: 599–605, 2013. © 2012 Wiley Periodicals, Inc.

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