Banca de DEFESA: PIETRA BRUNA BARBOSA BENTO

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : PIETRA BRUNA BARBOSA BENTO
DATA : 22/08/2025
HORA: 09:00
LOCAL: Sala de conferência 601-3, Bloco A, Campus Santo André
TÍTULO:

Characterization and nano-immobilization of a novel thermophilic esterase belonging to the Thermotogaceae family with potential for biotechnological applications

PÁGINAS: 45
RESUMO:

Esterases are a class of enzymes that catalyze the hydrolysis of ester bonds, widely used in the food, pharmaceutical, and detergent industries. In this study, we present the isolation, biochemical and biophysical characterization, and nano-immobilization of a novel thermophilic esterase belonging to the Thermotogaceae family (named here Tbe). Based on this approach, we hypothesize that the presence of the esterase enzyme during the synthesis process from AgNO₃ enables the formation of the nanoparticles and their immobilization.The three-dimensional structure of the Tbe, modeled using the AlphaFold program, exhibited a classical α/β-hydrolase fold formed by eight β-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. Tbe was cloned, expressed in E. coli, and purified to near homogeneity using affinity chromatography and size exclusion chromatography. The secondary structure was analyzed by circular dichroism spectroscopy, revealing a predominantly α-helical secondary structure for recombinant Tbe, as suggested by molecular modeling. The melting temperature value determined for Tbe at pH 8 was of 78 ± 1°C, revealing the thermophilic behavior of the enzyme. Tbe was able to hydrolyze p-nitrophenyl acetate (pNPA), exhibiting a specific activity of 25,814.6 U/mg, confirming its esterase activity, and reaching its optimal activity at pH 8.0 and 50 °C. The kinetic parameters determined for the hydrolysis of pNPA indicated a Km of 1.66 µmol/min, a kcat of 143.2 s⁻¹, and a Vmax of 4.29 µmol/min. X-ray diffraction (XRD) analysis revealed that the synthesized nanoparticles exhibited characteristic diffraction planes of both silver chloride (AgCl) and metallic silver (Ag⁰), evidencing the formation of a composite structure of the Ag@AgCl type. Based on this result, we report for the first time the successful photochemical synthesis of stable and functional nanoparticles formed from AgNO₃ solution and stabilized by the enzyme Tbe (named here as Tbe-Ag/AgCl NPs). The nanoparticles displayed various morphologies, including spherical and cubic shapes, with crystalline nature and heterogeneous diameters ranging from 20 to 200 nm. Our findings showed that the esterase activity of Tbe was maintained in the Tbe-Ag/AgCl NPs. Finally, we observed a significant increase in the thermostability of Tbe when associated with the nanoparticles. The information described here should provide a useful basis for future research and biotechnological applications of nano-immobilized esterases.

MEMBROS DA BANCA:
Presidente - Interno ao Programa - 1763495 - WANIUS JOSE GARCIA DA SILVA
Membro Titular - Examinador(a) Interno ao Programa - 3292123 - DANILO TRABUCO DO AMARAL
Membro Titular - Examinador(a) Externo à Instituição - ANA LUIZA DA ROCHA FORTES SARAIVA - EMBRAPA
Membro Suplente - Examinador(a) Interno ao Programa - 1601025 - MARCELLA PECORA MILAZZOTTO
Membro Suplente - Examinador(a) Externo à Instituição - MATHEUS LOPES SILVA