Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process.

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000 g·mol-1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2 mL·min-1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.

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)

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573 / Production and characterization of fibrinolytic protease from Streptomyces parvulus DPUA 1573

As proteases fibrinolíticas são capazes de degradar coágulos de fibrina formados dentro dos vasos sanguíneos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrução jugular e também um edema de laringe, derivando a obstrução das vias aéreas, o que possibilita um edema cerebral, ocorrendo o óbito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessária a investigação de outras fontesde proteases fibrinolíticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinolíticas obtidas de Streptomyces parvulus DPUA 1573. Para produção da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentração da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37ºC) e agitação (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolítica. Pode-se verificar que a protease fibrinolítica apresentou atividade máxima (835U/mL) nas condições de concentração de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentação. A protease fibrinolítica obtida teve temperatura e pH ótimos de 55°C e pH 9,0, respectivamente. A atividade enzimática foi inibida pelo EDTA, pelo íon Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolítica, possuindo características bioquímicas favoráveis à aplicação na medicina veterinária e possivelmente humana.(AU)

Fibrinolytic proteases are able to degrade fibrin clot formed in the blood vessel, avoiding intravascular thrombosis. In animals, thrombophlebitis often affects horses, and in severe cases causes obstruction of the jugular and laryngeal edema leading to airway obstruction allowing cerebral edema resulting in the death of the animal. Since treatment is costly, the investigation of other sources of fibrinolytic proteases at lower cost and with fewer side effects is needed. Thus, this study aims to produce and characterize fibrinolytic proteases from Streptomyces parvulus DPUA 1573. For enzyme production, a factorial design was performed to evaluate 24 soybean flour concentration (0.5, 1.0 and 1.5%) and glucose (0, 0.5 and 1.0g/L), temperature (28, 32 and 37°C) and agitation (150, 200 and 250rpm) on biomass and fibrinolytic activity. Fibrinolytic protease showed maximum activity (835 U/mL) under these conditions: 1.5% soybean flour, 1g/L glucose, 28°C, and 150rpm 48 hours of fermentation. The optimal temperature was 55°C and optimal pH was 9.0. Fibrinolytic protease activity was inhibited by EDTA, the ion Fe2+, and by SDS, which indicated that the enzyme is a metallo-protease. The strain Streptomyces parvulus DPUA 1573 was able to produce fibrinolytic protease with biochemical characteristics favorable for application in veterinary and human medicine.(AU)

Produção de biocompostos com atividade antimicrobiana de Streptomyces sp. ante isolados de mastite caprina / Production of biocompounds with antimicrobial activity of Streptomyces sp. Isolated in face of goat mastitis

Actinomicetos são um dos principais produtores de enzimas, vitaminas e metabólitos secundários, destacando-se o gênero Streptomyces, o qual tem uma ampla capacidade de produção de antibióticos eficazes no combate a diferentes microrganismos, entre eles o Staphylococcus sp. Em virtude dessa eficiência no combate a patógenos, o objetivo deste trabalho foi avaliar a produção de metabólitos com atividade antimicrobiana produzidos por 67 Streptomyces isolados de liquens da região amazônica, ante isolados de mastite caprina (Staphylococcus aureus) do estado de Pernambuco, Brasil. Foi utilizado um planejamento fatorial para avaliar a influência das fontes de carbono (glicose) 0%, 0,5% e 1% e de nitrogênio (farinha de soja) 1%, 2,5% e 4% na produção dos antimicrobianos, bem como das variáveis pH, biomassa e atividade antimicrobiana. Dos Streptomyces estudados, o DPUA 1566 foi o que se destacou por formação de halos de inibição entre 18 e 26mm ante os isolados de mastite caprina. Foi possível verificar que a fonte de carbono inibiu a produção de antimicrobianos quando submetidos a uma concentração de glicose de 1%; com a retirada desta, os Streptomyces apresentaram uma elevada capacidade de produção de metabólitos com atividade antimicrobiana tendo potencial para o tratamento de mastite caprina.

Actinomycetes are a leading producer of enzymes, vitamins and secondary metabolites, especially the genus Streptomyces, which have a large capacity for the production of natural antibiotics and are effective against various micro-organisms including Staphylococcus sp. Due to this efficiency in combating micro-organisms, the aim of this study was to evaluate the production of metabolites with antimicrobial activity produced by Streptomyces isolated from lichens 67 in the Amazonia, compared to isolates from goat mastitis (Staphylococcus aureus) in the state of Pernambuco, Brazil. We used a complete factorial design to evaluate the influence of concentrations of carbon sources (glucose) at 0%, 0.5% and 1% and nitrogen (soybean flour) at 1%, 2.5% and 4% in the production of antimicrobial metabolites, and the influence of the pH, microbial biomass and activity variables. Of the studied Streptomyces DPUA 1566 what stood out was the formation of inhibition halos between 18 to 26 mm compared to the isolates from goats mastitis. It was noted that the carbon source inhibited the production of antimicrobial metabolites when subjected to a glucose concentration of 1%. However, after the discontinuation, Streptomyces showed a high capacity to produce metabolites with antimicrobial activity, which has an excellent potential for the treatment of mastitis in goats.

Case reports: hyperbaric oxygen therapy for the treatment of cerebral air embolism.

Cerebral air embolism is one of the most deleterious disorders that may affect divers, but it is also a possible complication of surgeries and medical procedures. We report our experience with iatrogenic cerebral air embolism and hyperbaric oxygen treatment.

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.

Extraction of ascorbate oxidase from Cucurbita maxima by continuous process in perforated rotating disc contactor using aqueous two-phase systems.

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Situação epidemiológica da brucelose bovina no Estado de Minas Gerais / Epidemiological status of bovine brucellosis in the State of Minas Gerais, Brazil

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose bovina no Estado de Minas Gerais. O Estado foi estratificado em sete circuitos produtores. Em cada circuito foram amostradas aleatoriamente cerca de 300 propriedades e, dentro dessas, foi escolhido, de forma aleatória, um número pré-estabelecido de animais, dos quais foi obtida uma amostra de sangue. No total, foram amostrados 20.643 animais, provenientes de 2.204 propriedades. Em cada propriedade visitada aplicou-se um questionário epidemiológico para verificar o tipo de exploração e as práticas zootécnicas e sanitárias que poderiam estar associadas ao risco de infecção pela doença. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e a confirmação dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo, se pelo menos um animal foi reagente às duas provas sorológicas. As prevalências de focos e de animais infectados do Estado foram de 6,0 por cento [5,0-7,1 por cento] e 1,1 por cento [0,78-1,4 por cento], respectivamente. Os resultados para os circuitos pecuários da prevalência de focos e de animais foram: circuito 1, 4,7 por cento [2,7-7,7 por cento] e 0,82 por cento [0,06-1,6 por cento]; circuito 2, 7,2 por cento [4,6-10,6 por cento] e 1,2 por cento [0,53-1,8 por cento]; circuito 3, 6,8 por cento [4,3-10,0 por cento] e 1,5 por cento [0,47-2,4 por cento]; circuito 4, 6,5 por cento [4,1-9,8 por cento] e 1,1 por cento [0,39-1,7 por cento]; circuito 5, 3,8 por cento [2,0-6,5 por cento] e 0,40 por cento [0,11-0,69 por cento]; circuito 6, 6,2 por cento [3,8-9,6 por cento] e 0,66 por cento [0,29-1,0 por cento]; circuito 7, 11,0 por cento [7,7-15,0 por cento] e 1,7 por cento [0,92-2,6 por cento], respectivamente. Os fatores de risco (odds ratio, OR) associados à condição de foco foram: compra de reprodutores (OR = 1,66 [1,13-2,44]), ocorrência de aborto nos últimos 12 meses (OR = 1,81 [1,26-2,60]) ...

A study to characterize the epidemiological status of brucellosis was carried out in the State of Minas Gerais. The State was divided in seven regions. Three hundred herds were randomly sampled in each region and a pre-established number of animals was sampled in each of these herds. A total of 20,643 serum samples from 2,204 herds were collected. In each herd, it was applied an epidemiological questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT), and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME). The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalence of infected herds and animals in the State were, respectively, 6.0 percent [5.0-7.1 percent] and 1.1 percent [0.78-1.4 percent]. In the productive regions, the prevalence of infected herds and animals were, respectively: regions 1, 4.7 percent [2.7-7.7 percent] and 0.82 percent [0.06-1.6 percent]; region 2, 7.2 percent [4.6-10.6 percent] and 1.2 percent [0.53-1.8 percent]; region 3, 6.8 percent [4.3-10.0 percent] and 1.5 percent [0.47-2.4 percent]; region 4, 6.5 percent [4.1-9.8 percent] and 1.1 percent [0.39-1.7 percent]; region 5, 3.8 percent [2.0-6.5 percent] and 0.40 percent [0.11-0.69 percent]; region 6, 6.2 percent [3.8-9.6 percent] and 0.66 percent [0.29-1.0 percent]; and region 7, 11.0 percent [7.7-15.0 percent] and 1.7 percent [0.92-2.6 percent]. The risk factors (odds ratio, OR) associated with positive herds were: purchase of breeding stock (OR = 1.66 [1.13-2.44]), occurrence of abortions over the last 12 months (OR = 1.81 [1.26-2.60]), and the presence of deer in the farm (OR = 1.56 [1.08-2.27]). Vaccination against brucellosis was a protective factor (OR = 0.38 [0.19-0.79]). It can be concluded that the compulsory vaccination ...

Hypoglycaemia due to insulin autoimmune syndrome: report of two cases with characterisation of HLA alleles and insulin autoantibodies.

OBJECTIVE: Insulin autoimmune syndrome (IAS) has been reported mainly in Japan and so far only 27 IAS cases have been described from outside Asia. We describe two unrelated Portuguese patients with IAS and characterise their insulin autoantibodies and HLA alleles. PATIENTS: Patient 1, a 24-year-old white female suffered an episode of unconsciousness in the late postprandial state and blood glucose was found to be 33 mg/dl with serum insulin levels of >3980 microIU/ml (normal range 0-30 microIU/ml). She was receiving monthly injections of penicillin G for the prophylaxis of recurrent tonsillitis. Patient 2, was a 19-year-old white female, with episodes of sweating, hand tremor, weakness and hunger occurring in the postprandial state and blood glucose levels during the attacks of 28-56 mg/dl. Very high insulin levels (602-708 microIU/ml) were present. METHODS AND RESULTS: Anti-insulin antibodies, determined by a semi-quantitative method, were strongly positive in both patients (91.7% in patient 1 and 88.6% in patient 2; normal range < or =7%). Sephadex G-100 chromatography of the sera showed most of insulin immunoreactivity present in the void volume which was retained by an affinity column with anti-human-immunoglobulin G antibodies (87% and 95% from patients 1 and 2 respectively). Scatchard plot analysis and molecular typing of the DRB1 gene revealed a polyclonal antibody and DRB1*0406 in patient 1, and a monoclonal antibody and DRB1*0403 in patient 2. CONCLUSIONS: These two Portuguese patients with IAS had different HLA-DR4 subtypes and insulin autoantibodies: DRB1*0406 and a polyclonal antibody in a patient treated with penicillin, and DRB1*0403 and a monoclonal antibody in a patient with "idiopathic" IAS.