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Recombinant Laccase: I. Enzyme cloning and characterization
Author(s) -
Nicolini Claudio,
Bruzzese Debora,
Cambria Maria Teresa,
Bragazzi Nicola Luigi,
Pechkova Eugenia
Publication year - 2013
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.24397
Subject(s) - laccase , recombinant dna , microbiology and biotechnology , escherichia coli , lac operon , biochemistry , complementary dna , chemistry , enzyme , expression vector , molecular cloning , biology , gene
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.