Enhancement of the biological activity of hydroxytyrosol through its oxidation by laccase from Trametes versicolor.

The laccase-catalyzed oxidation of hydroxytyrosol (HT) towards the formation of its bioactive oligomer derivatives was investigated. The biocatalytic oligomerization was catalyzed by laccase from Trametes versicolor in aqueous or various water-miscible organic solvents and deep eutectic solvent (DES)-based media. Mass Spectroscopy and Nuclear Magnetic Resonance were used for the characterization of the products. The solvent system used significantly affects the degree of HT oligomerization. The use of 50 % v/v methanol favored the production of the HT dimer, while other organic solvents as well as DESs led to the formation of hydroxytyrosol trimer and other oligomers. In vitro studies showed that the HT dimer exhibits 3- to 4-fold enhanced antibacterial activity against Gram-positive and Gram-negative bacteria compared to the parent compound. Moreover, the ability of HT dimer to inhibit the activity of soybean lipoxygenase and Candida rugosa lipase was 1.5-fold higher than HT, while molecular docking supported these results. Furthermore, HT dimer showed reduced cytotoxicity against HEK293 cells and exhibited a strong ability to inhibit ROS formation. The enhanced bioactivity of HT dimer indicates that this compound could be considered for use in cosmetics, skin-care products, and nutraceuticals.

Tyrosinase Magnetic Cross-Linked Enzyme Aggregates: Biocatalytic Study in Deep Eutectic Solvent Aqueous Solutions.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at -20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media.

In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from Candida antarctica (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance.