title: Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea.
abstract: Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Tonin
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy. Electronic address: elena.rosini@uninsubria.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Rosini
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Piubelli
authors.affiliation: Department of Genetics and Microbiology, University of Murcia, Murcia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Sanchez-Amat
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism; D053544:Marinomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002396:Catechols; D011994:Recombinant Proteins; D008031:Lignin; D004156:Catechol Oxidase; D042845:Laccase; C034221:catechol
keywords: Laccase; Lignin degradation; Protein solubilization; Recombinant protein