Biochemical and Structural Characterization, and Nano-Immobilization of an Esterase from a Thermotogaceae Bacterium Active on Terephthalate Esters

The substantial amounts of polyethylene terephthalate (PET) plastic being released and accumulating in the ecosystem pose a significant challenge due to its recalcitrant structure. PET, widely used in packaging, textiles, and consumer goods, is highly resistant to natural degradation processes, leading to persistent environmental pollution. The use of thermophilic enzymes in the degradation of materials such as PET offers an alternative for breaking down this material, particularly due to their ability to operate under extreme temperature conditions. In this study, we cloned, expressed and purified a thermophilic enzyme from a Thermotogaceae bacterium named here TbE. The three-dimensional structure of the TbE was modeled using the AlphaFold program. Crystals of the TbE were obtained and will be diffracted on the Sirius X-ray line (Synchrotron, Campinas, Brazil) soon. Preliminary biochemical characterization using p-nitrophenyl acetate (pNPA) indicated optimal activity at pH 8. Temperature effects were evaluated and showed high activity at 50°C. The TbE displayed activity against pNPA, p-nitrophenyl butyrate, p-nitrophenyl decanoate, bis(2-hydroxyethyl) terephthalic acid (BHET), dimethyl terephthalate (DMT) and mono-4-nitrophenyl terephthalate (MpNPT). We also obtained results regarding the activity against others esters substrates, such as: phenyl propionate, octyl acetate, vinyl butyrate, vinyl propionate, γ-valerolactone, tripropionin, geranyl acetate, ciclohexyl butyrate, glyceryl tributyrate, glyceryl triacetate, butyl acetate, and diethyl phthalate. We also evaluated the effects of temperature in the conformational structure by circular dichroism spectroscopy. Also, TbE immobilization was performed via the formation and stabilization of silver chloride nanoparticles (TbE/AgCl-NPs). The TbE/AgCl-NPs showed activity in pNPA even after immobilization. This study represents a step forward in developing novel enzymes with potential biotechnological applications for terephthalate esters degradation. 

This work was supported by CAPES (88882.461674/2019-01), FAPESP (2020/15595-3; 2022/08958-8) and CNPq (307199/2023-1).