Aumento da estabilidade da thimet-oligopeptidase (EC 3.24.15) imobilizada em nanopartículas de ouro (THOP- GNP)

• Ano de defesa: 2023
• Autor(a) principal: Ramos, Marcos Paulo Cyrillo [UNIFESP]
• Orientador(a): Oliveira, Vitor [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo
• Palavras-chave em Português: Thimet-oligopeptidase; Nanopartícula de ouro; Protease; Atividade enzimática
• Palavras-chave em Inglês: Gold nanoparticle; Enzymatic activity
• Link de acesso: https://repositorio.unifesp.br/11600/67444

Objective: The development of biotechnological techniques that allow the use of proteases for disease treatment are an expanding field. However, the low stability of proteases is a limiting factor, and thus, an efficient strategy would be the immobilization of proteases such as THOP in gold nanoparticles (GNPs) for possible therapies. Methodology: The study protein was purified and characterized by chromatography and circular dichroism spectroscopy, thus demonstrating a pure protein (more than 90% purity) and with an adequate structure. Then, the synthesis of THOP-GNPs was carried out and their subsequent characterization by spectroscopy (UV-vis), proteolytic activity and atomic force microscopy. Results: The study protein was complexed with the nanoparticle and stability was observed, retaining 91.9% of its THOP-GNP activity synthesized with protein at 4 nM for 6h. The complexed enzyme resulted in retention of specificity by MALDI-TOF mass spectrometry, incubating the enzyme complexed to GNPs next to its natural substrate BK, maintaining the hydrolysis profile of its substrate, between the results of phenylalanine and serine, when it obtained the free enzyme. Conclusion: The results obtained from this work demonstrate that the characterization, synthesis and complexation of THOP in GNP can help us to standardize and apply this process with other biologically active proteins. When associated with GNPs, THOP presents high stability, potentially useful as an application in therapies when there is a need for BK processing.