title: A novel, simple screening method for investigating the properties of lignin oxidative activity.
abstract: Enzymatic lignin degradation represents a key challenge for integrated biorefineries. Notwithstanding the rich content in aromatic compounds, lignin's complex structure has hampered identification of an effective and cost-efficient enzymatic procedure to transform it into less complex product families. Advancements in enzymatically modifying or degrading lignin require a simple and reliable analytical method to quickly screen diverse lignin samples by employing different enzymes and conditions. Here, we report on a novel, rapid, and economic colorimetric assay for lignin oxidation based on the reaction of 2,4-dinitrophenylhydrazine with the carbonyl groups generated by enzymatic oxidation. The assay was validated on monomeric and dimeric lignin model compounds by comparison with HPLC analysis. The colorimetric method was used to investigate the activity of ten laccases and eight peroxidases on three technical lignins under different experimental conditions (e.g., by altering pH and mediator used). The colorimetric method was also coupled to a size-exclusion chromatographic separation of the lignin sample obtained after the enzymatic treatment in order to verify whether the enzymatic treatment resulted in lignin depolymerization, too. On the basis of this novel procedure, appropriate enzymatic treatments can now be identified to generate valuable lignin product streams.
authors.affiliation: Department of Biotechnology and Life Sciences, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, via Mancinelli 7, 20131, Milano, Italy; Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133, Milano, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Tonin
authors.affiliation: Department of Biotechnology and Life Sciences, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Vignali
authors.affiliation: Department of Biotechnology and Life Sciences, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, via Mancinelli 7, 20131, Milano, Italy.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.affiliation: The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, via Mancinelli 7, 20131, Milano, Italy; Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, p.zza Leonardo da Vinci 32, 20133, Milano, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: D'Arrigo
authors.affiliation: Department of Biotechnology and Life Sciences, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy; The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, via Mancinelli 7, 20131, Milano, Italy. Electronic address: elena.rosini@uninsubria.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Rosini
mesh_terms: D001709:Biotechnology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry / Q000379:methods*; D057166:High-Throughput Screening Assays; D042845:Laccase / Q000378:metabolism; D008031:Lignin / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D008031:Lignin; D042845:Laccase; D010544:Peroxidases; C042858:lignin peroxidase
keywords: Colorimetric assay; Lignin oxidation; Lignin valorization; Ligninolytic enzymes; Screening method