Development of novel biocatalysts and biosystems for green chemistry

The chemical synthesis currently employed in the manufacture of cosmetics presents limitations such as unwanted side reactions and the need of strong chemical conditions. In order to overcome these drawbacks, novel enzymes have been developed to catalyse the targeted bioconversions contributing to the solution of the environmental concerns of the industrial activities moving them towards sustainable biotechnologies. This work was mainly aimed at developing improved biocatalysts based on feruloyl esterases (FAEs) and glucuronoyl esterases (GEs) for the production of compounds with antioxidant activity. Novel fungal FAEs and GEs, identified through a bioinformatics approach from the analysis of 300 fungal genomes by the “Westerdijk Fungal Biodiversity Institute”, were expressed in Pichia pastoris and characterized. FAE from Aspergillus wentii was selected as the most promising enzyme to be subjected to site-directed mutagenesis to further fine-tune the enzyme towards its application in bioconversions. A homology model of this enzyme was developed and five site-directed variants were designed, expressed in P. pastoris and characterized assessing substrate specificity, solvent and thermo tolerance. This analysis led to the development of a rational designed variant with tenfold improved hydrolytic activity and a variant with enhanced thermo and solvent tolerance. As a second approach to develop improved biocatalysts based on FAEs, directed evolution was applied to the already characterized FAEs from Fusarium oxysporum (FoFaeC) and from Myceliophthora thermophila (MtFae1a). Two complete methodologies for the construction and the automated screening of evolved variants collections were developed and applied to the generation of 30,000 mutants libraries and their screening. Randomly mutated variants of FoFaeC and MtFae1a were generated through error prone-polymerase chain reaction and expressed in Yarrowia lipolytica and Saccharomyces cerevisiae, respectively. Thanks to the development of ad hoc chromogenic substrates for high-throughput assays on solid and in liquid media, screening for higher extracellular FAE activity than the wild type enzymes led to the selection of improved enzyme variants. The best evolved variants of both MtFae1a and FoFaeC were characterized for their thermotolerance, solvent tolerance and specificity towards methylated cinnamic substrates and subjected to small molecular docking studies to assess substrate interactions. In addition, MtFae1a evolved variants were tested in transesterification reactions in detergentless microemulsions for the production of target compounds selected for their potential antioxidant activity. Thus, although the screening strategy was based on the selection of evolved variants with improved hydrolytic activity, it was possible to obtain MtFae1a variants with both hydrolytic and synthetic enhanced activities to be potentially exploited in cosmetic industry. Finally, with the aim of identifying novel FAEs with non-conserved sequences, different fungal strains isolated from lignocellulosic biomasses during biodegradation under natural conditions and belonging to the microbial collection of Department of Agriculture (University of Naples “Federico II”) were screened for the production of different enzymes having potentially synergistic actions on lignocellulose conversion. This led to the selection of a novel fungal species showing FAE activity production induced by different carbon sources. Genome and transcriptome sequencing and analysis confirmed the presence of genes related to plant cell wall degrading enzymes.