Journal of Cellular Biochemistry 2013-03-01

Recombinant laccase: I. Enzyme cloning and characterization.

Claudio Nicolini, Debora Bruzzese, Maria Teresa Cambria, Nicola Luigi Bragazzi, Eugenia Pechkova

Index: J. Cell. Biochem. 114(3) , 599-605, (2013)

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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.Copyright © 2012 Wiley Periodicals, Inc.