Dottorato in SCIENZE CHIMICHE

Titolo della tesi: A new challenge in the bioeconomy: agro-industrial wastes as cheap and novel carriers for laccase immobilization

Biocatalysis, by cells or enzymes, is today a consolidated technique in biotechnological uses, and represents a crucial strategy in sustainable or green chemistry as a consequence of the reduced process time, intake of low energy input, non-toxicity and for an extraordinary chemo-, regio- and enantio-selectivity. The use of biocatalysts and their ever-greater diffusion is mainly due also to important technological advances. In recent years, the use of free enzymes in industrial process has increased, but with some limitations such as instability, recovery inability and the high costs. Protein engineering, coupled with the possibility of immobilizing enzymes on suitable supports, may allow overcoming the stability limitation under different conditions. Among the supports, agro-industrial wastes have been increasingly studied thanks to their high availability, high surface area, no toxicity, cheapness, low resistance to mass transfer, and good accessibility of the catalyst to the substrate. However, they have been little explored as a support for enzymatic immobilization. Thus, the aim of this research was to study the immobilization of laccase from Trametes Versicolor on three different agro-industrial wastes (eggshell membrane, spent grain and rice husk) as a strategy to improve the enzyme catalytic efficiency and stability. The first step was to characterize the chosen waste to understand its composition, size, and morphology. Then, a systematic study of laccase immobilization on eggshell membrane, spent grain and rice husk, was carried out. On this purpose, the type of interaction (chemical or physical) between support and enzyme, the changes in enzymatic activity profile after immobilization, and reuse of the proposed biocatalysts, were investigated. In details, the thesis is divided in three main sections: i) The choice of a suitable carrier for laccase immobilization considering the potentiality, characteristics, and composition (with or without cellulose) of the most common used wastes in biocatalysis reported in literature (paper I);ii) Laccase immobilization on not lignocellulosic wastes: eggshell membrane (paper II) with two different types of enzyme loadings: by membrane soaking in laccase solution or by enzyme-dropping on membrane. From the obtained results appeared that for the soaking procedure the immobilization of periodate-oxidized laccase on NiCl2-pretreated eggshell membrane was the best method (immobilized activity 1300 U/Kg, a residual activity of 30 % for 6 reuse). For the enzyme-dropping protocol, the covalent immobilization with the bifunctional cross linker (glutaraldehyde) was the optimal one (immobilized activity 3500 U/Kg, a residual activity of 45 % for 6 reuse) and finally the system was able to remove syringic acid from an aqueous solution. iii) Laccase immobilization on two different lignocellulosic wastes: spent grain (paper III and IV) and rice husk (paper V). The effect of spent grain delignification on immobilization was firstly analyzed in paper III. The procedure was fundamental to obtain a better exposure of cellulose for the laccase physical adsorption. The optimized conditions reported in paper III were: delignification with H2SO4/NaOH (it guaranteed an immobilized activity five times higher than HCl/NaOH procedure) and physical dropping as immobilization procedure (2500 U/Kg vs 1100 U/Kg obtained with the dropping covalent procedure). The biocatalyst also had good reusability (42% of activity retained after four cycles), and good catalytic efficiency (0.053 min-1). To implement the performance of the biocatalyst and make it suitable for continuous application (paper IV), the spent grain was shredded, delignified with H2SO4/NaOH and used for the laccase physical immobilization at pH 3. A packed bed reactor was then filled with the biocatalyst. The system showed an higher reusability (80% of activity retained after three cycles) and catalytic efficiency (0.16 min-1) than the biocatalyst proposed in paper III. The system was then tested for the decolorization of three aqueous solutions dyes both in batch mode and continuous recirculation by HPLC pump of the sample solution. The system showed best performance in the dyes removal in recirculation mode (for example 70% for methyl orange after 120 min) and good reusability (70% for 7 repeated cycles). Lastly, mesoporous nanosilica was obtained from rice husk (paper V) and characterized by XRD, FT-ATR, SEM and EDX. The XRD patterns revealed the amorphous nature of the extracted silica, EDX confirmed the presence of pure SiO2, while FT-ATR analysis showed the existence of silanol and siloxane groups. Silica was introduced in an alginate-chitosan scaffold and after the amination with APTES, the three-dimensional material was used for laccase immobilization to allow for greater ease to use. Results show that silica improved thermal and mechanical properties of the scaffold and the biocatalyst was able to totally oxidize syringic acid in 24 h. The all results obtained showed the potential of agro-industrial waste to act as a support for enzyme immobilization, creating biocatalysts with promising industrial applications, which can improve processes and make them more sustainable. In this way, the waste recycling allows to reduce environmental issues and to take full advantage of the immobilization benefits.