Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and arabinose residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D). The use of FAEs as accessory enzymes for the degradation of lignocellulosic biomass and their synergism with other hemicellulases has been studied for application in many industries, such as the food, the biofuel and the paper pulp industry. In the recent years, the use of FAEs as biosynthetic tools has been underlined. Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters resulting in compounds with altered lipophilicity, revealing a great potential for tailor-made modification of natural antioxidants for use in cosmetic, cosmeceutical and pharmaceutical industries.

The first part of the thesis is focused on the optimization of reaction conditions for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate using 5 FAEs (FaeA1, FaeA2, FaeB1, FaeB2 and MtFae1a) from Myceliophthora thermophila in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation. Regarding the synthesis of prenyl ferulate, FaeB2 offered the highest transesterification yield (71.5±0.2%) after 24 h of incubation at 30^oC using 60 mM vinyl ferulate (VFA), 1 M prenol and 0.02 mg FAE/mL in a mixture comprising of 53.4: 43.4: 3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0. At these conditions, the competitive hydrolysis was 4-7 fold minimized. Regarding the synthesis of L-arabinose ferulate, FaeA1 offered highest transesterification yield (35.9±2.96%) after 8 h of incubation at 50^oC using 80 mM VFA, 55 mM L-arabinose and 0.02 mg FAE/mL in a mixture of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. It was revealed that the type B FAEs from M. thermophila show higher preference to more lipophilic acceptors like prenol, while the type A FaeA1 was more efficient in the synthesis of the more hydrophilic L-arabinose ferulate.

The second part of the thesis is focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger (AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamate esters. For this purpose, L-arabinose ferulate and caffeate were synthesized enzymatically. kcat/Km ratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification.

Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.

Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.

Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.

In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.

Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.