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.

Prototypes of Newly Conceived Inorganic and Biological Sensors for Health and Environmental Applications

“…A LB thin film [20] of recombinant laccase from Rigidoporus lignosus (formerly known as Rigidoporus microporus ), obtained as previously reported [6], was prepared using a highly concentrated sample of laccase. The mixed chloroform solution in equimolar proportions had a concentration of 1 mg/mL.…”

“…We used inorganic nanocomposites, such as matrices of calcium oxide [2], and biological ones such as Nucleic Acid Programmable Protein Arrays (NAPPA) [3,4] along with Langmuir-Blodgett (LB) multi-layers of proteins of primary interest such as laccase [5,6]. Special attention was paid to the detection of substances significant for both the environment (such as carbon dioxide) and medicine (drugs and cancer) by means of a wide variety of detection methods (amperometric, conductometric, and nanogravimetric).…”

“…The enzyme implemented in our device [5] was laccase [6], which is a blue oxidase capable of oxidizing phenols and aromatic amines by reducing molecular oxygen to water by means of a full complement of copper atoms. Laccase belong to a large group of multicopper enzymes, which includes among others ascorbic acid oxidase and ceruloplasmine.…”

Ab Initio Semi-Quantitative Analysis of Micro-Beam Grazing-Incidence Small-Angle X-Ray Scattering (Μ-GISAXS) during Protein Crystal Nucleation and Growth

“…LB nanobiotemplate enables scientists to finely manipulate molecules and proteins, and allows the development and design of highly ordered nanobiopatterns and sensitive nanobiosensors [15][16][17].…”

Why P6 medicine needs clinical psychology and a trans-cultural approach

“…In the last decades, medicine has undergone profound and deep changes and challenges, such as the introduction of new medical devices and nanobiotechnologies, [1][2][3] that have enabled the understanding of molecular pathogenesis at unprecedented levels. Nanogenomics and nanoproteomics represent the frontiers of biomedicine, [4][5][6][7] paving the way for a personalized diagnosis and treatment, that is to say not just a one-size-fits-it-all therapy as before but a tailored, targeted therapeutics.…”