Use and characterisation of free or immobilised enzymatic systems for the synthesis and functionalisation of novel materials

The work reported in this Ph.D. thesis is focused on the use and immobilisation of enzymes to produce new materials, which are important for biotechnological applications. The use of enzymes in industrial sectors is continuously increasing. Enzymes offer many advantages over traditional chemical processes. The research work of this thesis can be divided into two parts. The first part is focused on the immobilisation of laccase and chitinase. The main object of enzymatic immobilisation is to enhance the economics of biocatalytic processes. Enzymatic immobilisation allows the reuse of enzymes for an extended period of time and enables easier separation of the catalyst from the product. Furthermore, immobilisation improves many properties of enzymes: performance in the organic solvents, pH tolerance and heat stability. The most widely used immobilisation method is the covalent binding of the enzyme to support. Different type of support can be chosen for enzymatic immobilisation. As the material can plays a crucial role in the immobilisation process and the properties of the produced catalytic system. In this thesis we have chosen two different supports: super paramagnetic nanoparticles for both the enzymes used and the chitosan beads as an alternative support for chitinase. Magnetic nanoparticles show interesting properties for enzymatic immobilisation, they can be obtained with small size, increasing the yield of enzymatic immobilisation and above all, the reaction products can be easily recovered applying an external magnetic field. Magnetic nanoparticles were prepared following the traditional method of co-precipitation of Fe2+ and Fe3+ ions. This immobilisation process was successfully used for chitinase, obtaining a high immobilisation yield and increasing enzymatic stability. Different was for laccase, which having a different catalytic mechanism a revision of the synthesis has been attempt. The use of the magnetic nanoparticles obtained with the traditional method hampered the detection of stable radical species formed during the catalytic mechanism as it happens for the oxidation product of 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), the standard compound used to test the enzyme activity. Changing some synthetic parameters, the new magnetic nanoparticles were produced and characterized. In fact nanoparticles with a lower aggregation state and a smaller hydrodynamic diameter were obtained and tested without any interference with the ABTS substrate. Chitinase was also immobilised on chitosan beads/Macro-Spheres (CMS), as this support is completely atoxic and so most suitable for application in food industries.The presence of active amino groups in deacetylated GlcNAc units of chitosan also enables the binding of the linker (glutaraldehyde and genipin) and then of the enzyme. The goal of this part of the thesis was to attempt the immobilisation of Chitinase on different supports, MNPs and CMS, for the efficient production of COS. The second part of this thesis is focused on the use of enzymes to produce melanin pigments. Melanins have a variety of biological functions, including protection against UV radiation, free radical scavengers and metal ions chelators. Thanks to their properties, melanins found applications in several fields such as cosmetics, optoelectronics, food, and pharmacology. Eumelanin and Pheomelanin have been produced by oxidative enzymatic synthesis using laccase from Trametes versicolor and then characterized by the use of Multifrequency Continuos Wave (CW) and pulse Q-band EPR. Then, as soluble melanin pigments have important technological applications in different fields, like in optoelectonics, soluble pigments mimicking pyomelanin structure have been synthesized starting from Homogentisic Acid and Gentisic Acid monomers and spectroscopically characterized with their antioxidant activities determination.