Development of an enzymatic tool-box for lignin oxidation/degradation. Sviluppo di un tool-box enzimatico per l’ossidazione e la degradazione della lignina.

Lignin is an amorphous polymer characterized by a wide range of molecular mass components, a disordered and branched three-dimensional structure, insoluble in water and in most common solvents. In order to perform lignin degradation, enzymatic treatment could represent an environmentally friendly alternative to chemical methods. The main purpose of this PhD project was to develop an "enzymatic tool-box" for an efficient oxidation and degradation of lignin into aromatic monomers. Biochemical properties of available commercial and recombinant ligninolytic enzymes (laccase, Mn peroxidase and lignin peroxidase) were evaluated under identical experimental conditions, with the final goal to identify interesting biocatalysts for lignin degradation. The effect of pH, temperature, NaCl, DMSO and Tween-80 on the enzymatic activity has been investigated. The activity of novel enzymes, such as the membrane-bound polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) and a peroxidase produced by Nonomuraea gerenzanensis, was also evaluated. A new high-throughput colorimetric screening to assay the oxidation/degradation of lignin by different enzymes was developed: this method facilitates the identification of optimal conditions for a lignin treatment based on the combined use of various laccases and peroxidases. On this side, coupling the colorimetric assay with a size-exclusion chromatography analysis allows to identify changes in lignin molecular mass distribution due to enzymatic treatment. Finally, based on recent literature, a chemo-enzymatic process to depolymerise lignin was carried out on two lignin linkage model compounds and on four technical lignins. This could represent an innovative and feasible way for valorisation of lignin under mild conditions.