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Proteases da biomassa microalgal de Chlorococcum infusionum: obtenção, caracterizações físico-químicas e enzimáticas

Mon, 01 Jan 2024 00:00:00 GMT

Título: Proteases da biomassa microalgal de Chlorococcum infusionum: obtenção, caracterizações físico-químicas e enzimáticas Autor(es): Ferreira, Vanessa Rodrigues Abstract: The biodiversity of microalgae remains a relatively underexplored resource. Its biomass represents a rich source of natural bioactive compounds, such as proteins and lipids, which hold potential applications in human and animal nutrition, as well as in the cosmetic, pharmaceutical, and fine chemical industries. Moreover, they exhibit promise in wastewater treatment and the bioremediation of harmful compounds. Specifically, microalgae belonging to the Chlorococcum genus are considered promising sources of macromolecules; however, their potential as protein sources remains inadequately investigated. In this context, the objective of this study was to extract, isolate, and evaluate the physicochemical and enzymatic characteristics of proteases present in the biomass of Chlorococcum infusionum microalgae produced in a photobioreactor. To achieve this, the microalgae were initially cultivated in Bold's Basal Medium (BBM) in an autotrophic closed-flat plate photobioreactor system. The resulting biomass was characterized and utilized in the preparation of aqueous extracts through a combination of lysis methods (EBCi). The microalgae cultivation yielded an average fresh biomass of 2.35 ± 0.03 g.L-1 after the 12th day of cultivation, with a composition rich in lipids (37.82%) and proteins (27.32%). For the isolation of proteases, EBCi underwent purification steps involving subsequent gel filtration chromatography using a Sephadex G-100 column and ion exchange chromatography using a DEAE-Sepharose™ Fast Flow column. The predominant peak obtained (designated as FPCiT-P3) was subjected to proteolytic activity assays against a nonspecific substrate (azocasein) and through zymograms. Optimal parameter determination assays, stability assays against temperature and pH variations, and structural stability evaluation through circular dichroism spectroscopy were conducted. FPCiT-P3 exhibited an optimum pH for activity around 7.0-9.0 and an optimum temperature of 50°C, being partially inhibited by PMSF and EDTA. The fraction showed low stability at acidic pHs and temperatures above 60°C after a 30-minute incubation. The circular dichroism spectrum of FPCiT-P3 indicated a predominance of β-sheet structure. Tipo: Dissertação

Toxicidade de nanopartículas de prata em modelos aquáticos e a interferência do meio na agregação da nanopartícula

Thu, 01 Jan 2026 00:00:00 GMT

Título: Toxicidade de nanopartículas de prata em modelos aquáticos e a interferência do meio na agregação da nanopartícula Autor(es): Martins, Alexia Riquet Abstract: Silver nanoparticles (AgNP) are of high industrial interest, standing out for their antiseptic and antifungal properties and versatility in drug delivery. However, the expansion of their employability is directly linked to the increase in the disposal of these nanoparticles in the environment. Worldwide, regulations such as REACH and the EPA exist, although not specifically targeted, which cover the use, production, and disposal of nanoparticles. In Brazil, Bill 880/2019 (Legal Framework for Nanotechnology) stands out. Regardless of regulation, risk assessment of a nanomaterial to be used on a large scale is a basic requirement to ensure the safety of use. Aiming to standardize toxicity and experimental parameters, the OECD establishes standards, guidelines, and study models for testing materials. Among the model organisms for studies in aquatic environments and food chains are the microalgae Chlamydomonas reinhardtii, the microcrustacean Artemia salina, and Danio rerio embryos. These organisms were chosen for testing exposure to AgNP concentrations (12.5, 25, and 50 ppm) and testing the trophic level passage of nanoparticles through their synthesis, characterization of AgNP, and analysis of damage caused to the models using microscopy. The AgNP used was synthesized in collaboration and analyzed in UV-Vis, with two syntheses and one standard, and ten (10) times more concentrated; the exposure experiments in the three models were carried out by diluting the synthesized AgNP in the respective model media with analyses every 24 hours. The trophic level experiment consisted of exposing C. reinhardtii previously contaminated with AgNP concentrations to A. salina (nauplii I and II), and at the end of the experiments, the samples were collected and fixed for optical, scanning electron, and confocal microscopy. The results obtained showed that in the A. salinas medium, colloids tend to form, visible in the digestive tract of the specimen without causing morphological changes or mortality. In the homemade TAP medium, AgNP does not show the same tendency to aggregate, in addition to decreasing the replication speed of C. reinhardtii, which form colonies surrounded by a transparent secretion. D. rerio embryos showed 100% mortality, and the trophic level experiment showed no toxicity caused by the ingestion of C. reinhardtii contaminated by A. Salina. It is concluded that the exposure medium directly influences the toxicity of AgNP, which proved to be toxic when kept in its non-aggregated state. Tipo: Dissertação

Efeito dos revestimentos à base de carragenana extraída de Hypnea pseudomusciformis na vida útil de filés de salmão (Salmo salar) refrigerado.

Wed, 01 Jan 2025 00:00:00 GMT

Título: Efeito dos revestimentos à base de carragenana extraída de Hypnea pseudomusciformis na vida útil de filés de salmão (Salmo salar) refrigerado. Autor(es): Silva, Aline Almeida da Abstract: The preservation of refrigerated fish is an economic and sanitary challenge due to its high susceptibility to microbial growth and chemical reactions that impair sensory and nutritional quality. Edible coatings based on marine polysaccharides have been proposed as physical barriers and delivery systems for preservative agents. Among them, carrageenan extracted from red algae stands out for its availability, biocompatibility, and good film-forming properties. This study evaluated the effect of carrageenan-based edible coatings, extracted from the macroalga Hypnea pseudomusciformis, on the shelf life and quality of salmon fillets (Salmo salar) stored at 0 °C for 18 days. The carrageenan was obtained by aqueous extraction (steps at 25 °C and 80 °C), ethanol precipitation, dialysis, and lyophilization, and the polymer solutions were prepared at concentrations of 1.0, 1.5, and 2.0% (w/v), with and without Tween 80. Preliminary wettability assays (spreading coefficient Ws) guided the selection of the applied formulation. The 1.0% (w/v) solution without surfactant showed the best balance between viscosity, spreading ability, and repeatability, and was selected for application to the fillets. FTIR analyses and surface tension measurements were performed. During storage, pH, TVB-N, TMA-N, TBARS, total aerobic count (TPC), and K-value by HPLC were monitored. The coating delayed nucleotide degradation kinetics, reducing the increase in K-value by ~25% and keeping the fillets within the moderate freshness range (K < 40%) for 15 days, compared to 12 days in the control. In parallel, TPC in coated fillets remained below 7.0 log10 CFU/g until day 15, while the control exceeded this limit on day 12, indicating a 3-day extension in microbiological shelf life. FTIR spectroscopy confirmed the high purity and reproducibility of the extracted carrageenan. These results support the potential of H. pseudomusciformis carrageenan as a promising physical barrier for the preservation of refrigerated fish. Tipo: Tese

Desenvolvimento de um protocolo de simulação por dinâmica molecular para avaliação estrutural e energética de variantes da l-asparaginase humana

Wed, 01 Jan 2025 00:00:00 GMT

Título: Desenvolvimento de um protocolo de simulação por dinâmica molecular para avaliação estrutural e energética de variantes da l-asparaginase humana Autor(es): Silva, Juliana Meneses de Sena Abstract: L-asparaginase is an enzyme widely employed in the treatment of acute lymphoblastic leukemia (ALL) due to its ability to degrade L-asparagine (Asn), which is essential for the survival of leukemic cells. However, currently available formulations are derived from bacterial sources and are often associated with severe immune reactions. Although the human body possesses a homologous enzyme, hASNase1, its low catalytic activity precludes its direct clinical application. To overcome this limitation, mutations have been proposed to enhance its catalytic efficiency. This study aimed to develop a computational protocol, based on molecular dynamics (MD) simulations and structural analyses, to evaluate the structural and energetic effects of hASNase1 variants and to identify molecular features associated with increased catalytic activity. The systems were built based on the native structure of the enzyme and simulated under physiological conditions. Structural stability was assessed through root mean square deviation (RMSD), while interatomic interaction potentials (PII) and binding free energy calculations were used to analyze the interactions between hASNase1 variants and Asn. MM/PBSA calculations revealed increasing binding affinities in the order D87S < D84N < A186V < D87N, consistent with substrate residence times in the catalytic site. Experimentally, catalytic activity increases of 17× (D87S), 19× (D84N), 50× (A186V), and 52× (D87N) were observed compared to the wild-type enzyme, confirming the effectiveness of the DM + MM/PBSA protocol in identifying high-performance variants. In conclusion, the DM and MM/PBSA protocol elucidated the structural determinants underlying the experimentally observed activity gains in hASNase1 variants, identifying D87N and A186V as promising candidates for an optimized human L-asparaginase. Understanding the relationship between structure and catalytic activity in hASNase1 variants reinforces the use of in silico approaches in the rational development of safer and more effective biopharmaceuticals for the treatment of ALL. Tipo: Dissertação