Produção e purificação integrada de protease fibrinolítica de Mucor subtilissimus UCP 1262 / Integrated production and purification of fibrinolytic protease from Mucor subtilissimus UCP 1262

As enzimas fibrinolíticas podem ser obtidas de micro-organismos por meio de processos fermentativos. O presente trabalho teve como objetivo avaliar a produção e extração integrada da protease fibrinolítica de Mucor subtilissimus UCP 1262 usando sistema de duas fases aquosas (SDFA). O processo integrado foi realizado para avaliar a produção, partição e recuperação da protease fibrinolítica, segundo planejamento experimental 23, utilizando como variáveis independentes a massa molar do polietileno glicol (PEG), a concentração do PEG e a concentração do sulfato de sódio. A maior atividade fibrinolítica (15,40U/mL) foi obtida na fase rica em sulfato de sódio no ensaio composto por 10% de sal e 18% de PEG 8000 (g/mol). Recuperações superiores a 80% foram obtidas. A protease fibrinolítica apresentou pH ótimo 7,0, estabilidade entre os pH 6,0 e 8,5, temperatura ótima 50°C, sendo estável de 10°C a 50°C. A enzima foi classificada como uma serino protease, com massa molecular de 52kDa. Como resultado, o processo é notavelmente eficaz para pré-purificar a protease fibrinolítica com baixo custo e rapidez significativa. Quando comparada a outras técnicas de produção e purificação isoladas, a fermentação extrativa é um processo digno a ser substituto das etapas iniciais de separação convencionais.(AU)

Fibrinolytic enzymes can be obtained from microorganisms through fermentative processes. The study aimed to evaluate the fibrinolytic protease production and integrated extraction from Mucor subtilissimus UCP 1262 by extractive fermentation using Aqueous Two-Phase Systems (ATPS). The integrated process was carried out to assess the production, partition and fibrinolytic enzyme recovery, according to a 2 3 -experimental design, using as independent variables Polyethylene glycol (PEG) molar mass, PEG and sodium sulphate concentration, concentration. The highest fibrinolytic activity (15.40U/mL) was obtained in sodium sulfate rich phase in the assay comprising of 10% of salt and 18% of PEG 8000 (g/mol). Yield greater than 80% was obtained. The fibrinolytic protease presented optimum pH 7.0 and stability between pH 6.0 and 8.5, and optimum temperature 50°C, stable between 10°C to 50°C. The enzyme was classified as a serine-protease with 52kDa of molecular weight. As a result, the process is remarkably effective to pre-purify the fibrinolytic protease with a low cost and significantly faster processing time. When compared to other isolated production and purification techniques the extractive fermentation is worthy of being a candidate to replace the initial stages of conventional separation processes.(AU)

Synergistically enhanced stability of laccase immobilized on synthesized silver nanoparticles with water-soluble polymers.

Silver nanoparticles (AgNPs) were synthesized by citrate reduction method in the presence of polymers, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and chitosan, used as stabilizing agents, and an oxidoreductase enzyme, laccase (Lac), with the goal of expanding the NPs antimicrobial action. AgNPs were characterized by UV-vis spectrometry, dynamic light scattering and transmission electron microscopy. As protecting agents, PEG and PVA promoted the formation of spherical uniformly-shaped, small-sized, monodispersed AgNPs (≈20nm). High Mw polymers were established as most effective in producing small-sized NPs. Chitosan's viscosity led to the formation of aggregates. Despite the decrease in Lac activity registered for the hybrid formulation, AgNPs-polymer-Lac, a significant augment in stability over time (up to 13days, at 50°C) was observed. This novel formulation displays improved synergistic performance over AgNPs-Lac or polymer-Lac conjugates, since in the former the Lac activity becomes residual at the end of 3days. By enabling many ionic interactions, chitosan restricted the mass transfer between Lac and substrate and, thus, inhibited the enzymatic activity. These hybrid nanocomposites made up of inorganic NPs, organic polymers and immobilized antimicrobial oxidoreductive enzymes represent a new class of materials with improved synergistic performance. Moreover, the Lac and the AgNPs different antimicrobial action, both in time and mechanism, may also constitute a new alternative to reduce the probability of developing resistance-associated mutations.

Actinomicetos produtores de inibidores de beta-lactamases com atividade antimicrobiana frente a isolados de mastite bovina / Actinomycetes producers of beta-lactamases inhibitors with antimicrobial activity against microorganisms isolated from bovine mastitis

Avaliou-se a capacidade de 71 actinomicetos isolados de líquens da região amazônica em produzir inibidores de β-lactamases com atividade antimicrobiana sobre Staphylococcus aureus, resistentes à penicilina, isolados de mastite bovina do estado de Pernambuco. A seleção dos actinomicetos produtores de inibidores de β-lactamases foi realizada pela técnica de bloco de gelose contra Klebsiella pneumoniae ATCC 29665, e os actinomicetos selecionados foram testados frente a 17 linhagens de Staphylococcus aureus resistentes à penicilina. Os melhores produtores de inibidores de β-lactamases foram Streptomyces sp. DPUA 1542 e Nocardia sp. DPUA 1571, os quais foram submetidos ao cultivo submerso para determinação da curva de crescimento, pH e atividade antimicrobiana. Os maiores halos de inibição foram obtidos pelos metabólitos produzidos após 96 horas de cultivo tanto para Nocardia sp. - 13,5 e 12,0mm - como para Streptomyces sp. - 8,0 e 14,0mm - com os testes de difusão nos discos e poços, respectivamente. Os resultados permitiram concluir que os actinomicetos são fonte promissora de inibidores de β-lactamases, com potencial uso no tratamento de mastites bovinas.

The ability of 71 actinomycetes, isolated from the Amazon lichens, to produce β-lactamase inhibitors with antimicrobial activity was evaluated against penicillin-resistant Staphylococcus aureus, isolated from bovine mastitis in Pernambuco State. The selection of actinomycetes producers of β-lactamase inhibitors was performed using agar-plug method against Klebsiella pneumoniae ATCC 29665 and the selected actinomycetes were tested against 17 penicillin-resistant Staphylococcus aureus strains. The best producers of β-lactamase inhibitors were Streptomyces sp. DPUA 1542 and Nocardia sp. DPUA 1571. They were submitted to the submerged cultivation to determine the growth and pH curve, and antimicrobial activity. The highest inhibition halo zonewas obtained by metabolites produced after 96 hours of cultivation for both Nocardia sp. (13.5 and 12.0mm) and Streptomyces sp. (8.0 and 14.0mm) with discs and well diffusion tests, respectively. The results showed that the actinomycetes are a promising source of β-lactamase inhibitors, with potential for use in the bovine mastitis treatment.

Screening of variables influencing the clavulanic acid production by Streptomyces DAUFPE 3060 strain.

Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28 degrees C, 30 degrees C, and 32 degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32 degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

Parametros hematologicos normais em Fortaleza, Ceara. 1. Serie vermelha. / Normal hematological parameters in Fortaleza, Ceara. 1. Red blood cells

Determina os parametros hematologicos: hemacias, hemoglobulina, hematocrito, indices hematimetricos, reticulocitos e hemossedimentacao, em 200 individuos adultos normais, residentes em Fortaleza, Ceara.Os valores encontrados foram submetidos a analise estatisca e os resultados comparados com os da literatura