Purificação de toxinas produzidas por Clostridium perfringens: uma revisão

Clostridium perfringensé uma bactéria anaeróbia Gram-positiva, amplamente distribuída no meio ambiente e comumente encontrada no intestino de animais, incluindo o homem. As espécies de C. perfringensestão classificadas em cinco tipos toxigênicos (A, B, C, D, E) em função da produção de quatro toxinas (±, ², µ, ¹). Entretanto, as toxinas teta, delta, lambda e enterotoxina são também sintetizadas por outras espécies dessa bactéria. Muitas metodologias para purificação das toxinas produzidas por C. perfringens têm sido propostas e, portanto, nesta revisão foram apresentados e discutidos os métodos e resultados de purificação dessas toxinas relatados nas últimas quatro décadas.Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (±, ², µ, ¹). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades

Factorial design for collagenase production by Penicillium sp. selected from the Caatinga soil

Collagenolytic proteases can hydrolyze both denatured and native collagen and are becoming increasingly important commercially. The aim of this work was to select a new strain of Penicillium sp. isolated from the soil of Caatinga for collagenase production. A factorial design 24 was performed to determine the best conditions of enzyme production. Collagenolytic activity reported on this work is about 2 times larger than existing data. According to the growth kinetics, after 126 hours of production, were obtained the highest values of collagenolytic activity and specific activity. The highest values of collagenolytic activity and specific activity were obtained on a culture medium containing 0.25% (w/v) gelatin, 200 rpm, pH 8.0 and 24 °C. Only two factors were statistically significant: pH and temperature, both with negative effects. The experimental design made possible to define fermentation culture conditions able to increase by 66% the value of the initial enzyme activity.Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation the best condition of Fibrinolytic protease production using factorial design by Streptomyces sp DPUA 1573

XI Reunião Regional Nordeste da SBBq | 4th International Symposium in Biochemistry of Macromolecules and BiotechnologyFibrinolytic enzymes have the ability to degrade fibrin clots formed for avoiding intravascular thrombosis. In the pharmaceutical industry there is a search for new fibrinolytic agent that reduces the production cost and increasing productivity. The use of microorganism for enzyme production, such as the genus Streptomyces has been reported. Streptomyces is a Gram-positive bacteria, responsible for producing many bioactive compounds and extracellular enzymes of pharmaceutical interest. This study aimed to evaluated the production of fibrinolytic protease by Streptomyces sp DPUA 1573. Microbial cells were cultivated in the ISP2 for 48 hours, after this period the strains were inoculated in MS2 (soybean medium) that according with factorial design 24 (concentrations of soybean 0.5; 1.0 and 1.5%, glucose 0; 0.5 and 1.0% and different speeds 150 rpm; 200 rpm and 250 rpm and temperature 28C; 30C and 32C). The factorial design was analyzed by variance analysis (anova) and the glucose concentration showed a positive and significative effect. The results showed that the variable interaction had significant effect. that the best condition was composed 1.5% soybean, 1% glucose, 28 ºC and 150 speed in 48 hours, with production fibrinolytic 1391.66 U/mL. These values were higher than those reported in the literature. However these results show the biggest potencial in production fibrinolytic enzyme by Streptomyces.info:eu-repo/semantics/publishedVersio

Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle

The sugarcane borer, Diatraea saccharalis, is one of the worst pests in Brazilian sugarcane crop, causing high levels of financial losses every year. Beauveria bassiana is an entomopathogenic fungus widely used in the biological control of several agricultural pests. The aims of this study were to: (1) evaluate the pathogenicity of B. bassiana strains against D. saccharalis (2) investigate the production of proteases and chitinase by B. bassiana in the presence of the cuticle of sugarcane borer; and, (3) evaluate the relation between the production of enzymes and pathogenicity of the strains. All isolates tested were pathogenic to D. saccharalis and the mortality ranged from 36 to 88%. The production of enzymes was higher in the medium containing cuticle, showing that the process is stimulated by specific components found in the cuticle of the host. Pr1 activity was higher than Pr2 and both were produced at 24 h. The highest production of chitinase was obtained at 96 h of culture for all strains tested. Levels of specific cuticle-degrading enzymes such as proteases correlated positively with specific virulence parameters. B. bassiana URM2915 showed promising features to be used in a biological control program of D. saccharalis.Key words: Biological control, sugarcane, subtilisin-like protease, trypsin-like protease, chitinase

Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An in vitro evaluation of probiotic properties

The therapeutic use of probiotics for supporting the antibiotic action against gastrointestinal disorders is a current trend and emerging applications have gained popularity because of their support for various microbiological activities in digestive processes. Microorganisms isolated from kefir with great probiotic properties, in addition to high resistance to harsh environmental conditions, have been widely researched. Administration of probiotic yeasts offers a number of advantages, when compared to bacteria, because of particular characteristics as their larger cell size. In the present study, 28 strains of Saccharomyces cerevisiae were isolated, after in vitro digestion of kefir-fermented milk, and identified by molecular based approaches. A screening was performed to determine important quality requirements for probiotics including: antagonistic and antioxidant activities, -galactosidase synthesis, autoaggregation, surface hydrophobicity and adhesion to epithelial cells. The results showed strains: with antagonistic activity against microbial pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis; able to produce -galactosidase; with antioxidant activity levels higher than 90%; with hydrophobicity activity and autoaggregation ability (evaluated by adhesion test, where all the strains presented adhesion to mice ileal epithelial cells). These findings are relevant and the strains are recommended for further in vivo studies as well as for potential therapeutic applications.The authors thank the financial support of CAPES (National Council for the Improvement of Higher Education); the identification of yeast strains by Micoteca URM (Department of Mycology -UFPE); professor Daniela Marques and PhD student Rebeca Melo for providing the mice intestine and the laboratory of Histology (DMFA-UFRPE) for the histological pictures.info:eu-repo/semantics/publishedVersio

Avaliação de variáveis que influenciam a hidrólise enzimática da caseína do leite de cabra Moxotó

The aim of this work was to evaluate the effects of temperature, pH, enzyme:substrate ratio (E:S), and reaction time on the enzymatic hydrolysis of Moxotó breed goat's milk casein, using different proteolytic enzymes. Enzymatic hydrolysis of the capric casein was performed using trypsin, pepsin and papain. For each enzyme, two levels of the variables were used to assess their effects on the hydrolysis of casein, using a full factorial design (24). The hydrolysis products were visualized on SDS-PAGE. The best hydrolysis degree using papain were at pH 6.5, E:S of 1:150 and 5 hours of hydrolysis at 50°C (28.17%); for trypsin, at pH 8.5, E:S of 1:150 and 5 hours at 40°C (29.55%); and for pepsin, at pH 3.0, E:S of 1:100 and 5 hours at 40°C (38.27%). Capric casein hydrolysis is affected positively by pH and reaction time, using pepsin. Significant antagonistic interactions were observed between pH and E:S, with trypsine; and between pH, temperature and reaction time, with papain. Pepsin has high αs1, β e κ-casein hydrolysis efficiency, evidenced by the molar masses below 14.4 kDa of its products.O objetivo deste trabalho foi avaliar os efeitos da temperatura, pH, relação enzima:substrato (E:S) e tempo de reação sobre a hidrólise enzimática da caseína do leite de cabra Moxotó, com uso de diferentes enzimas proteolíticas. A hidrólise enzimática da caseína caprina foi realizada com uso de tripsina, pepsina e papaína. Para cada enzima, foram utilizados dois níveis de cada variável, na avaliação de seus efeitos sobre a hidrólise da caseína, em um fatorial completo 24. Os produtos da hidrólise foram visualizados em eletroforese SDS-PAGE. O melhor valor do grau de hidrólise com a enzima papaína foi obtido em pH 6,5, E:S de 1:150 e 5 horas de hidrólise a 50ºC (28,17%); com a tripsina, em pH 8,5, E:S de 1:150 e 5 horas a 40ºC (29,55%); e com a pepsina, em pH 3,0, E:S de 1:100 e 5 horas a 40ºC (38,27%). A hidrólise da caseína caprina é influenciada positivamente pelo pH e tempo de hidrólise, com o uso da pepsina. Foram observadas interações significativas antagônicas entre pH e relação E:S, com o uso da tripsina; e entre pH, temperatura e tempo de hidrólise, com o uso da papaína. A pepsina apresenta elevada eficiência na hidrólise de αs1, β e κ-caseína, evidenciada por hidrolisados com massa molar abaixo de 14,4 kDa

Production of enzymes by filamentous fungus using sugarcane and sugarcane bagasse as substrate

The production of enzymes by bioprocesses is a good alternative to add value to agroindustrial waste. Sugarcane bagasse, an abundant and cheap by-product of the sugar industry, was tested as a carbon source for the production of biotechnological interesting enzymes. In this work, fungi were isolated from anatomical parts of sugarcane (root, steam and leaf) and, then, were assessed for enzyme production. The isolated and identified fungi were Fusarium sp., Penicillium sp., Trichoderma auroviride and Cladosporium cladosporioides. Trichoderma auroviride was used for enzyme production (xylanase, invertase and protease) using sugarcane as substrate. Xylanase production (2037 U) by Trichoderma auroviride was higher than invertase and protease production; thus, this enzyme was selected for the further studies. The study of the influence of variables (temperature and stirring intensity) on xylanase production by Trichoderma auroviride, using sugarcane bagasse as substrate, showed that the most favorable xylanase production conditions were observed at 25 °C, without stirring intensity and using saline and Tween for enzyme extraction, which led to a 1980 U xylanase activity.(Produção de enzimas por fungos filamentosos utilizando cana-de-açúcar e bagaço de cana-de-açúcar como substrato). A produção de enzimas por bioprocessos é uma boa alternativa para agregar valor a resíduos agroindustriais. O bagaço da cana-de-açúcar, um abundante e barato subproduto da indústria de açúcar, foi testado como fonte de carbono para a produção de enzimas de interesse biotecnológico. Neste trabalho foi realizado o isolamento e identificação de fungos a partir de peças anatômicas (caule, raiz e folha) da cana-de-açúcar e em seguida foi realizada a investigação da produção de enzimas por esses microrganismos. Fusarium sp., Penicillium sp., Trichoderma auroviride e Cladosporium cladosporioides foram os fungos isolados e identificados. Trichoderma auroviride foi utilizado para a produção de enzimas (xilanase, invertase e protease) utilizando cana-de-açúcar como substrato. A produção de xilanase (2037 U) por Trichoderma auroviride foi maior que a produção de protease e de invertase, portanto, essa enzima foi selecionada para estudos posteriores. O estudo da influência das variáveis temperatura e intensidade de agitação na produção da xilanase por Trichoderma auroviride usando bagaço da cana-de-açúcar como substrato demonstrou que a condição mais favorável para a produção de xilanase foi observada a 25 °C, sem agitação e utilizando solução salina e Tween para extração da enzima, o que levou a uma produção de xilanase de 1980 U

The use of poultry feather to produce keratinase by Aspergillus carbonarius

O objetivo deste trabalho foi estudar a produção de queratinase por Aspergillus carbonarius URM 1546, tendo-se como substrato penas de galinha, por meio de planejamento fatorial completo 23. Todos os parâmetros estudados e as interações de segunda ordem foram estatisticamente significativos. A maior atividade queratinolítica (48,9 U mL-1) foi obtida com 120 rpm, 0,5% (p/v) de penas de galinha e sete dias de cultivo.The objective of this work was to evaluate the keratinase production by Aspergillus carbonarius URM 1546, using as substrate poultry feather in a full experimental design 23. The studied parameters and the second-order interactions were statistically significant. The maximum keratinase activity (48.9 U mL-1) was obtained using 120 rpm, with 0.5% (w/v) poultry feather and seven culture days

Do lixo para a indústria: recuperação de enzimas colagenolíticas obtidas a partir de resíduos intestinais de peixes para aplicação industrial/ From waste to the industry: recovery of collagenolytic enzymes obtained from fish intestinal residues for industrial application

O aumento da produção e consumo de peixes têm gerado uma grande quantidade de resíduos sólidos: ossos, pele, escamas e vísceras digestivas (intestino, fígado, estômago). Com o intuito de aproveitar esses subprodutos, são extraídas diversas biomoléculas, entre elas, a colagenase, enzima capaz de clivar o colágeno. O objetivo deste trabalho foi selecionar e extrair enzimas colagenolíticas a partir de resíduos de peixes como potencial para aplicação biotecnológica. O material biológico das espécies em estudo passou pelos processos de separação, maceração e homogeneização. O de robalo-flecha (Centropomus undecimalis) (102,41 ± 0,00 U/mg) foi a espécie que apresentou maior atividade colagenolítica dentre as demais espécies: anchova (Pomatomus saltatrix) (82,24 ± 0,00 U/mg), xixarro amarelo (Caranx bartholomaei (26,66 ± 0,00 U/mL), pampo (Trachinotus carolinus) (89,00 ± 0,07 U/mg), tambaqui (Colossoma macropomum) (78,23 ± 0,00 U/mg) e tilápia-do-Nilo Oreochromis niloticus (81,96 ± 0,01 U/mg). A partir desses resultados observou-se que o robalo-flecha (C. undecimalis) apresenta um possível potencial para aplicação biotecnológica, aumento do lucro das indústrias de pescados, além de reduzir o descarte inadequado ao meio ambiente