Feruloyl Esterases Protein Engineering to Enhance Their Performance as Biocatalysts: A Review.

Feruloyl esterases (FAEs) are versatile enzymes able to release hydroxycinnamic acids or synthesize their ester derivatives, both molecules with interesting biological activities such as: antioxidants, antifungals, antivirals, antifibrotic, anti-inflammatory, among others. The importance of these molecules in medicine, food or cosmetic industries provides FAEs with several biotechnological applications as key industrial biocatalysts. However, FAEs have some operational limitations that must be overcome, which can be addressed through different protein engineering approaches to enhance their thermal stability, catalytic efficiencies, and selectivity. This review aims to present a brief historical tour through the mutagenesis strategies employed to improve enzymes performance and analyze the current protein engineering strategies applied to FAEs as interesting biocatalysts. Finally, an outlook of the future of FAEs protein engineering approaches to achieve successful industrial biocatalysts is given.

Improved synthesis of the antifungal isobutyl o-coumarate catalyzed by the Aspergillus terreus type B feruloyl esterase

BACKGROUND Hydroxycinnamic acids and some of their derivatives are molecules with interesting biological activities; for instance, hydroxylated hydroxycinnamic esters have proved to have antifungal properties, and thus the generation of these molecules is of industrial importance. In this study, the direct esterification capacity of the pure recombinant type B feruloyl esterase from Aspergillus terreus (AtFAE B) was evaluated by its ability to catalyze the synthesis of isobutyl o-coumarate, an interesting antifungal molecule. A ternary solvent system (isooctane/isobutanol/water) was employed to improve the synthesis of isobutyl o-coumarate, assessing different substrate concentrations, enzyme load, water percentages and pH and temperature values. RESULTS AtFAE B showed the highest initial rate at 18% (v/v) isobutanol and 50 mM o-coumaric acid, 0.04 mg/ml of enzyme, 4% (v/v) water without buffer and 40C. AtFAE B half-lives at 30C, 40C and 50C were 16.5 h, 1.75 h and 3.5 min, respectively. Thus, we decided to evaluate the bioconversion yield at 30C, where the enzyme showed the highest operational stability. At this temperature, we obtained a yield of ~80% after only 8 h of reaction, using a 78:18:4 isooctane:isobutanol:water ternary solvent system, with 50 mM of o-coumaric acid.CONCLUSIONS Under these improved conditions, the productivity was 1.06 g isobutyl o-coumarate/L*h with a biocatalyst yield of 209.6 kg isobutyl o-coumarate/kg free AtFAE B, demonstrating the promising potential of AtFAE B to accept the non-canonical o-coumaric acid as the substrate and to achieve the synthesis of isobutyl o-coum