Navegando por Palavras-chave "Enzymatic activity"
Agora exibindo 1 - 3 de 3
Resultados por página
Opções de Ordenação
- ItemAcesso aberto (Open Access)Aumento da estabilidade da thimet-oligopeptidase (EC 3.24.15) imobilizada em nanopartículas de ouro (THOP- GNP)(Universidade Federal de São Paulo, 2023-04-14) Ramos, Marcos Paulo Cyrillo [UNIFESP]; Oliveira, Vitor [UNIFESP]; Icimoto, Marcelo Yudi [UNIFERSP]; http://lattes.cnpq.br/4421009566436244; http://lattes.cnpq.br/1476417303065197; https://lattes.cnpq.br/2390105077854601Objective: 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.
- ItemSomente MetadadadosEffect of pH and Temperature on the Activity of Enzymatic Extracts from Pineapple Peel(Springer, 2012-07-01) Coelho Silvestre, Marialice Pinto; Carreira, Raquel Linhares; Silva, Mauro Ramalho; Corgosinho, Flavia Campos [UNIFESP]; Pereira Monteiro, Marcia Regina; Morais, Harriman Aley; Universidade Federal de Minas Gerais (UFMG); Univ Fed Vales Jequitinhonha & Mucuri; Universidade Federal de São Paulo (UNIFESP)This study was carried out to characterize a crude extract from pineapple peel after precipitation by three methods with the aim of obtaining an enzymatic extract from agro-industrial waste. the characterization of these extracts involved the determination of both protein content and specific protease activities. the effects of pH and temperature on specific protease activity and on the stability of the extracts were also evaluated. the optimal values of specific activity for the crude extract (CE) were pH 6.0 (5.76 U mg(-1) protein) and 7.0 (5.71 U mg(-1) protein) and a temperature of 70 A degrees C (16 U mg(-1) protein). the average values for the relative specific activity were 17.4% (pH 3.0 to 9.0) and 42.7% (at 30, 50, and 70 A degrees C). the ethanolic extract had the highest specific activity (10.7 U mg(-1) protein) in comparison to the best results obtained for the isoelectric precipitation (7.7 U mg(-1) protein) and the ammonium sulfate precipitation (4.7 U mg(-1) protein). Moreover, the ethanolic extract was more stable than the CE, retaining 60.9% and 53.7% of the initial specific activity during the evaluation of the stability at different pH and temperature values, respectively. the optimal values of pH and temperature were almost the same for the crude and the ethanolic extracts. in addition, the ethanolic extract was more stable than the CE in the experimental conditions tested in this work.
- ItemSomente MetadadadosMitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates(Elsevier B.V., 2010-01-01) Marcondes, Marcelo F. [UNIFESP]; Torquato, Ricardo J. S. [UNIFESP]; Assis, Diego M. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Hayashi, Mirian A. F. [UNIFESP]; Oliveira, Vitor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (INIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system the IMP fusion protein, with a poly-l-lis-tag (6x His). was obtained by cloning the coding region of INIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia call BL21 (DE3) pLysS After isolation and purification of the fusion protein by affinity chromatography Using Ni-Sepharose resin, the protein was Purified further using ion exchange chromatography with a Hi-trap resource Q column the recombinant IMP was characterized by Western blotting using three distinct antibodies, circular dichioism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. the successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning. (C) 2009 Elsevier Inc All rights reserved.