Immobilization of E. coli expressing Bacillus pumilus CynD in three organic polymer matrices.

Cyanide is toxic to most living organisms. The toxicity of cyanide derives from its ability to inhibit the enzyme cytochrome C oxidase of the electronic transport chain. Despite its high toxicity, several industrial processes rely on the use of cyanide, and considerable amounts of industrial waste must be adequately treated before discharge. Biological treatments for the decontamination of cyanide waste include the use of microorganisms and enzymes. Regarding the use of enzymes, cyanide dihydratase (CynD), which catalyzes the conversion of cyanide into ammonia and formate, is an attractive candidate. Nevertheless, the main impediment to the effective use of this enzyme for the biodegradation of cyanide is the marked intolerance to the alkaline pH at which cyanide waste is kept. In this work, we explore the operational capabilities of whole E. coli cells overexpressing Bacillus pumilus CynD immobilized in three organic polymer matrices: chitosan, polyacrylamide, and agar. Remarkably, the immobilized cells on agar and polyacrylamide retained more than 80% activity even at pH 10 and displayed high reusability. Conversely, the cells immobilized on chitosan were not active. Finally, the suitability of the active complexes for the degradation of free cyanide from a solution derived from the gold processing industry was demonstrated.

Proteolytic system of Bacillus sp. CL18 is capable of extensive feather degradation and hydrolysis of diverse protein substrates.

1. Feathers are recalcitrant protein-rich wastes produced in huge amounts by poultry processing for meat production. Hence, feather bioconversion and protease production by Bacillus sp. CL18 were investigated. 2. Bacillus sp. CL18 demonstrated a remarkable feather-degrading potential. Through cultivations on feather broth (10 g l-1 feathers), 94.5% ± 3% of whole feathers were degraded after 4 d. Increases in soluble protein contents were observed and protease production was maximal also at d 4. This strain produced diverse proteolytic enzymes during growth. 3. Crude protease displayed optimal activity at 55°C (50-62°C), pH 8.0 (7.0-9.0) and a low thermal stability. Proteolytic activity increased in the presence of Ca2+, Mg2+, Triton X-100, Tween 20 and dimethyl sulphoxide. Inhibition profile indicated that crude protease contains, mainly, serine proteases. Enzyme preparation hydrolysed mainly casein and soy protein isolate. 4. The keratinolytic capacity of Bacillus sp. CL18 at moderate temperatures (30°C) might be appropriate for feather conversion, resulting in protein hydrolysates and proteolytic enzymes. Proteases are postulated to be added-value products that can be obtained from such a bioprocess.

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk / Microbiota termodúrica psicrotrófica proteolítica do leite cru refrigerado

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.(AU)

Os micro-organismos termodúricos são aqueles capazes de resistir ao processo de pasteurização do leite. Assim, a microbiota do leite pasteurizado é constituída por esses micro-organismos que podem influenciar diretamente na vida sua vida útil. O objetivo do presente trabalho foi identificar a microbiota termodurica psicrotrófica proteolítica do leite cru refrigerado. Foram avaliadas 20 amostras de leite cru refrigerado, plaqueadas e incubadas a 7°C por 10 dias. As cepas isolados foram repicadas em ágar leite para verificação da atividade proteolítica e inicialmente avaliadas microscopicamente e posteriormente identificadas por sequenciamento de DNA. Foi observado apenas um crescimento bacteriano e outros 10 isolados fúngicos termodúricos psicrotróficos em 40% das amostras avaliadas. Todos os isolados foram proteolíticos. O sequenciamento parcial do gene 16S rRNA da cepa bacteriana permitiu a identificação de Bacillus pumilus e a análise da região ITS1-5.8S-ITS2 dos isolados fúngicos os identificou como Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), e Geotrichum candidum (30%). Não foram encontrados estudos anteriores sobre a presença de B. pumilus no leite cru brasileiro. Considerando o potencial deteriorante de todos os isolados e presença de fungos termodúricos psicrotróficos no leite cru, é importante a continuidade trabalhos que avaliem sua resistência térmica e seu impacto na vida útil do leite pasteurizado, longa vida e derivados.(AU)

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk / Microbiota termodúrica psicrotrófica proteolítica do leite cru refrigerado

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.

Os micro-organismos termodúricos são aqueles capazes de resistir ao processo de pasteurização do leite. Assim, a microbiota do leite pasteurizado é constituída por esses micro-organismos que podem influenciar diretamente na vida sua vida útil. O objetivo do presente trabalho foi identificar a microbiota termodurica psicrotrófica proteolítica do leite cru refrigerado. Foram avaliadas 20 amostras de leite cru refrigerado, plaqueadas e incubadas a 7°C por 10 dias. As cepas isolados foram repicadas em ágar leite para verificação da atividade proteolítica e inicialmente avaliadas microscopicamente e posteriormente identificadas por sequenciamento de DNA. Foi observado apenas um crescimento bacteriano e outros 10 isolados fúngicos termodúricos psicrotróficos em 40% das amostras avaliadas. Todos os isolados foram proteolíticos. O sequenciamento parcial do gene 16S rRNA da cepa bacteriana permitiu a identificação de Bacillus pumilus e a análise da região ITS1-5.8S-ITS2 dos isolados fúngicos os identificou como Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), e Geotrichum candidum (30%). Não foram encontrados estudos anteriores sobre a presença de B. pumilus no leite cru brasileiro. Considerando o potencial deteriorante de todos os isolados e presença de fungos termodúricos psicrotróficos no leite cru, é importante a continuidade trabalhos que avaliem sua resistência térmica e seu impacto na vida útil do leite pasteurizado, longa vida e derivados.