title: Recombinant laccase: I. Enzyme cloning and characterization.
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.
authors.affiliation: Biophysics and Nanobiotechnology Laboratories, DIMES, University of Genoa, Genoa, Italy. cnicolini@ibf.unige.it
authors.forename: Claudio
authors.initials: C
authors.lastname: Nicolini
authors.forename: Debora
authors.initials: D
authors.lastname: Bruzzese
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Cambria
authors.forename: Nicola Luigi
authors.initials: NL
authors.lastname: Bragazzi
authors.forename: Eugenia
authors.initials: E
authors.lastname: Pechkova
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D055453:Coriolaceae / Q000201:enzymology* / Q000235:genetics; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005784:Gene Amplification; D006835:Hydrazones / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000235:genetics; D007763:Lac Operon; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D006835:Hydrazones; D011994:Recombinant Proteins; C009370:syringaldazine; D007544:Isopropyl Thiogalactoside; D042845:Laccase