RESUMEN
Abstract Bacteriocin has been identified as an excellent alternative to chemical preservatives due to its astonishing antimicrobial activity against food spoiling and food-borne pathogens. So there is a need to identify the newer and potent sources of bacteriocin producers. This study aims the isolation of potent bacteriocin producing microorganism from fresh fruits and vegetables, its production, purification, and characterization. Firstly, 43 isolates were analysed for its antimicrobial potential, out of which7 were found to inhibit the growth of various pathogens. Considering the results of antimicrobial activity; the microorganism isolated from mango was regarded as the most potent one; which was identified as Bacillus subtilis VS.70% ammonium sulphate precipitated and dialysed bacteriocin was purified using DEAE cellulose and sephadex G75 chromatography. Bacteriocin was purified by 24.64 fold with 8.65% recovery and its molecular weight was found to be 31.2kDa. The Purified bacteriocin was found to be stable at broad pH and temperature. It was found to be degraded by various proteases studied confirming its proteinaceous nature. Considering all these attributes; the purified bacteriocin isolated from Bacillus subtilis VS can be exploited by various food industries.
Asunto(s)
Péptido Hidrolasas/análisis , Bacteriocinas/análisis , Antiinfecciosos/análisis , Bacillus subtilis , CromatografíaRESUMEN
Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.
Asunto(s)
Agentes de Coagulación , Péptido Hidrolasas/análisis , Pleurotus/química , Queso/análisisRESUMEN
Abstract Introduction Candida albicans is the yeast most commonly affecting the oral cavity, sometimes causing infection. However, several factors may be associated with the onset of candidiasis, which may be related not only to the hygiene and health of individuals, but also to the pathogenicity of these microorganisms. Objective To evaluate the virulence factors of Candida yeasts isolated from the oral mucosa of elderly people living in the "Comunidade Lago do Limão", municipality of Iranduba, Amazonas state, Brazil. Material and method Testes were performed to assess the production of urease, proteinase, phospholipase and hemolysin. Statistical analysis used the Fisher's exact test and the Chi-squared test. Result Prevalence of non-albicans species was observed. As for virulence factors, all isolates were negative ureases, and there was prevalence of very strong proteinase production, whereas most isolates did not produce this enzyme in the phospholipase test. All yeasts analyzed presented hemolysin production, with grade IV hemolysis as the most prevalent. There was no statistically significant difference between the virulence of isolates from the oral cavity and the prostheses of the elderly analyzed. Conclusion Several virulence factors may present with high intensity in the presence of oral microbiota changes. In addition, non-albicans species present number of virulence factors similar to that of C. albicans, with high pathogenicity. This study allows a better analysis of candidiasis prevention strategies aiming to promote improvement in the health and quality of life for the elderly.
Resumo Introdução A Candida albicans é a levedura que mais acomete a cavidade oral, podendo causar infecção. Porém diversos fatores podem estar associados ao aparecimento da candidíase, que podem estar relacionados com a higiene e saúde dos indivíduos, mas também com a patogenicidade destes microrganismos. Objetivo Avaliar os fatores de virulência de leveduras do gênero Candida isoladas da mucosa oral dos idosos residentes na Comunidade Lago do Limão - Iranduba - Amazonas - Brasil. Material e método Foram realizados os testes de urease, proteinase, fosfolipase, e avaliação da produção de hemólise. Na análise estatística utilizou-se teste Exato de Fisher e Quiquadrado. Resultado Obteve-se a prevalência de espécies não-albicans, quanto aos fatores de virulência, todos os isolados foram ureases negativos, houve prevalência de produção muito forte de proteinase, enquanto que no teste da fosfolipase, a maioria dos isolados não apresentou produção desta enzima; todas as leveduras analisadas apresentaram produção de hemolisina, sendo mais prevalente a hemólise grau IV. Não houve diferença estatisticamente significativa entre a virulência dos isolados oriundos da cavidade oral e da prótese dos idosos analisados. Conclusão Diversos fatores de virulência podem apresentar-se com alta intensidade na presença de alterações da microbiota oral. Além disso, as espécies não-albicans apresentam fatores de virulência tanto quanto a C. albicans, com graus de patogenicidade elevados. Este estudo permite a análise de estratégias de prevenção da candidíase, com intuito de promover melhor saúde e qualidade de vida para os idosos.
Asunto(s)
Humanos , Anciano , Candida , Infecciones Relacionadas con Prótesis , Factores de Virulencia , Mucosa Bucal/fisiopatología , Péptido Hidrolasas/análisis , Brasil , Candida albicans , Grupos de PoblaciónRESUMEN
The aim was to evaluate the microbiological, chemical- physical, and shelf-life quality of milk samples after pasteurization (HTST) for 10 days or ultra-high temperature (UHT) treatment for 120 days. Raw milk counts of mesophilic aerobic microorganisms, Staphylococcus spp. and thermotolerant coliforms before HTST and UHT processing were 6.73 and 7.77; 2.84 and 4.30, and 4.68 and 4.37log10, respectively. Pseudomonas spp. were found in raw milk samples. No presence of any other microorganisms studied was detected and no microbial inhibitor was found. Processed samples met microbiological legal requirements. However, aerobic mesophilic counts for HTST pasteurized milk samples stored for 5 and 10 days increased to values comparable to those in raw milk. Composition chemical- physical of all samples were within legal limits. These results demonstrate that, although HTST and UHT processed milk comply with the microbiological standards required by Brazilian law, high microbial counts in raw milk are an issue, possibly due to failures in the early stages of the production chain. Increase in casein macropeptide (CMP), probably because of proteases psychrotrophic bacteria. It is concluded that the quality of raw milk directly influences the progressive increase of the CMP values.(AU)
O objetivo da presente pesquisa foi avaliar a qualidade microbiológica, fisco-química e a vida de prateleira de amostras de leite, após o processo de pasteurização rápida (HTST) ou de ultra-alta temperatura (UHT) durante 10 dias, ou de ultra-alta temperatura (UHT) por 120 dias. As contagens de micro-organismos aeróbios mesófilos, Staphylococcus spp. e de coliformes termotolerantes do leite cru utilizado para tratamentos HTST e UHT foram, respectivamente (log10): 6,73 e 7,77; 2,84 e 4,30 e 4,68 e 4,37. Foi constatada a presença de Pseudomonas spp. no leite cru. Não foi detectada a presença de nenhum outro micro-organismo estudado, e as amostras estavam isentas de inibidores microbianos. Após a pasteurização, todas as amostras apresentaram contagens microbianas compatíveis com os limites legais. No entanto, as amostras de leite pasteurizado apresentaram contagens de aeróbios mesófilos semelhantes ao leite cru após cinco e 10 dias de armazenamento. A composição físico-química de todas as amostras estava de acordo com os limites legais. Observou-se acréscimo dos níveis de caseinomacropeptídeo (CMP) no leite UHT, provavelmente em função das proteases de bactérias psicrotróficas. Conclui-se que a qualidade do leite cru influencia diretamente os valores de CMP.(AU)
Asunto(s)
Leche/química , Leche/microbiología , Péptido Hidrolasas/análisis , Caseína Quinasas/análisisRESUMEN
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)
Asunto(s)
Fermentación , Fibrinolíticos , Péptido Hidrolasas/análisis , Streptomyces , MetaloproteasasRESUMEN
Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Enfermedades Periodontales/microbiología , Candida albicans/aislamiento & purificación , Candida albicans/patogenicidad , Enfermedades de la Pulpa Dental/microbiología , Diabetes Mellitus Tipo 2/microbiología , Oxidación-Reducción , Péptido Hidrolasas/análisis , Enfermedades Periodontales/fisiopatología , Enfermedades Periodontales/diagnóstico por imagen , Bolsa Periodontal/microbiología , Fosfolipasas/análisis , Virulencia , ADN de Hongos , Radiografía Dental , Estudios de Casos y Controles , Reacción en Cadena de la Polimerasa , Estadísticas no Paramétricas , Cavidad Pulpar/microbiología , Enfermedades de la Pulpa Dental/fisiopatología , Enfermedades de la Pulpa Dental/diagnóstico por imagen , Diabetes Mellitus Tipo 2/fisiopatología , Electroforesis , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)
Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)
Asunto(s)
Animales , Dípteros , Larva/fisiología , Péptido Hidrolasas/análisis , Serina Proteasas , Electroforesis en Gel de Poliacrilamida , Miasis/veterinaria , Inhibidores de ProteasasRESUMEN
Background. The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen (AU)
Antecedentes. La vacuola de Saccharomyces cerevisiae está involucrada activamente en el mecanismo de autofagia, desarrollando una labor importante en la homeostasis, degradación, recambio proteico, desintoxicación y protección de la célula en condiciones de estrés. Por el contrario, las proteasas vacuolares de Candida glabrata aún no han sido estudiadas por completo. Objetivos. El presente trabajo describe por primera vez la actividad proteolítica vacuolar en C. glabrata. Métodos. Los estudios bioquímicos realizados en C. glabrata pusieron de manifiesto la presencia de diferentes actividades proteolíticas: aspartil proteinasa ácida, que actúa sobre sustratos como la albúmina y la hemoglobina ácida desnaturalizada; serín proteasa neutra, con actividad sobre el substrato de tipo colágeno hide powder azure, y serín carboxipeptidasa, que actúa sobre N-benzoil-tyr-pNa. Resultados. La obtención de una fracción subcelular mostró una elevada actividad enzimática específica de las tres proteasas, lo que permitió confirmar su localización vacuolar. Se realizaron análisis de la expresión de los genes CgPEP4 (CgAPR1), CgPRB1 y CgCPY1 (CgPRC1), codificantes de las actividades proteolíticas aspartil proteasa A, proteasa neutra B y carboxipeptidasa Y, respectivamente. Los resultados reflejan una regulación diferencial de la expresión de la proteasa, dependiendo de la fuente de nitrógeno. Conclusiones. Las proteasas codificadas por los genes CgPEP4, CgPRB1 y CgCPY1 podrían participar en el proceso de autofagia y supervivencia de este patógeno oportunista (AU)
Asunto(s)
Péptido Hidrolasas/análisis , Candida glabrata , Candida glabrata/aislamiento & purificación , Candida glabrata/patogenicidad , Carboxipeptidasas/análisis , Carboxipeptidasas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/patogenicidad , Vacuolas/virología , Candida glabrata/enzimología , Proteasas de Ácido Aspártico/análisis , Proteasas de Ácido Aspártico/aislamiento & purificación , Autofagia , Homeostasis , Benzoilarginina-Nitroanilida/análisis , Infecciones Oportunistas/microbiología , Vacuolas , Vacuolas/microbiología , Vacuolas/patologíaRESUMEN
Background: cases of superficial and invasive mycoses caused by emerging species of Candida have been increasingly reported over the last thirty years. The production of hydrolytic enzymes plays a central role in the fungal infective process. In Candida infections the secretion of both proteases and phospholipases are well-known virulence attributes. Aims: to determine the protease and phospholipase production from 58 human clinical isolates of Candida obtained from individuals with cutaneous candidiasis seen in the Human and Veterinary Diagnostic Mycology Sector from Universidade Federal Fluminense (UFF), Brazil, from November 2008 to August 2009. Methods: fungal identification was performed using biochemical tests. Proteolytic activity was detected on agar plates containing bovine serum albumin, and phospholipase production was determined on egg-yolk plates. Results: the Candida species isolated were Candida parapsilosis (27.59%), Candida famata (18.96%), Candida albicans (15.52%), Candida haemulonii (12.06%), Candida ciferri (8.62%), Candida guilliermondii (6.90%), Candida tropicalis (5.17%) and Candida lipolytica (5.17%). All isolates of C. albicans produced both protease and phospholipase. As regards the isolates of non-C. albicans Candida species, 53.06% and 4.08% were able to produce protease and phospholipase, respectively. For example, the majority of isolates of C. parapsilosis (15/16) produced protease, while 40% of C. ciferri isolates (2/5) were phospholipase producers. This study shows, for the first time, that C. ciferri and C. haemulonii strains were able to produce protease. Conclusions: collectively, our results showed that different species of Candida isolated from cutaneous lesions were able to produce proteases and/or phospholipases, which are multifunctional molecules directly involved in the infectious process of these fungi
Antecedentes: Los casos de micosis superficiales e invasoras relacionados con las especies emergentes de Candida se han reportado progresivamente durante las últimas tres décadas. La producción de enzimas hidrolíticas juega un papel central en varios contextos de la patogenicidad fúngica. Con respecto a la infección por Candida, la secreción de proteasas y fosfolipasas son atributos de virulencia bien conocidos. Objetivos: Determinar y comparar la producción de proteasa y fosfolipasa de 58 aislamientos clínicos humanos de diferentes especies de Candida obtenidos de pacientes con candidiasis cutánea, atendidos en el Sector de Diagnóstico Micológico Humano y Veterinario de la Universidad Federal Fluminense (UFF), durante el período de noviembre de 2008 a agosto de 2009. Métodos: La identificación de las especies de Candida se realizó mediante pruebas bioquímicas, la actividad proteolítica se detectó en placas de agar que contenían albúmina de suero bovino y la actividad fosfolipasa se determinó utilizando el método de la placa de yema de huevo semi-cuantitativa. Resultados: Las especies aisladas fueron Candida parapsilosis (27,59%), Candida famata (18,96%), Candida albicans (15.52%), Candida haemulonii(12,06%), Candida ciferri(8,62%), Candida guilliermondii(6,90%), Candida tropicalis (5,17%) y Candida lipolytica (5,17%). Todos los aislamientos de C. albicans produjeron tanto proteasa como fosfolipasa. El 53,06% de los aislamientos de Candida no-C. albicans fueron capaces de producir proteasa y el 4,08% fosfolipasa. La mayoría de los aislamientos de C. parapsilosis (15/16) produjo proteasa, mientras que el 40% de los aislamientos de C. ciferri (2/5) fueron productores de fosfolipasa. Se describe por primera vez en la literatura científica la producción de proteasas por cepas de C. haemulonii y C. ciferri. Conclusiones: Nuestros resultados muestran el potencial que tienen los aislamientos de Candida provenientes de lesiones cutáneas para producir proteasas y fosfolipasas (AU)
Asunto(s)
Humanos , Candidiasis Cutánea/microbiología , Candida/patogenicidad , Fosfolipasas/análisis , Péptido Hidrolasas/análisis , Candida/enzimología , Factores de VirulenciaRESUMEN
In the family of Euphorbiaceae, the genera Euphorbia and Sapium are known to contain essentially latex-bearing species. In the present study, the latex of Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil., and Sapium glandulosum (L.) Morong, plants native from Brazil, were examined concerning proteolytic activity. All studied species have proteins with significant proteolytic activity and E. papillosa has the greatest specific activity. Aiming to verify the type of protease present, an assay with different inhibitors was performed. In the three tested plants, the proteolytic activity was significantly inhibited by a serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). Using techniques of electrophoresis with polyacrylamide gels (SDS-PAGE), the subunits of proteins were separated according to their molecular masses, and the protein activity was visually detected by zymography.
Dentro da família Euphorbiaceae, os gêneros Euphorbia e Sapium são conhecidos por incluírem basicamente espécies produtoras de látex. No presente estudo, o látex das plantas Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil. e Sapium glandulosum (L.) Morong, espécies nativas do Brasil, foi analisado em relação à atividade proteolítica. Todas as amostras analisadas possuem proteínas com significativa atividade, sendo que o látex da espécie E. papillosa apresenta a maior atividade específica. Com o objetivo de analisar quais os tipos de proteases responsáveis pela atividade proteolítica, realizaram-se ensaios com diferentes inibidores. Nas três plantas testadas a atividade foi inibida significativamente pelo cloridrato de 4-(fluoreto de 2-aminoetilbenzenossulfonil) (AEBSF), um inibidor de serino-proteases. Utilizando técnicas de eletroforese em gel de poliacrilamida (SDS-PAGE), as subunidades das proteínas foram separadas de acordo com sua massa molecular e, através da zimografia, a atividade proteolítica pode ser detectada visualmente.
Asunto(s)
Péptido Hidrolasas/análisis , Euphorbiaceae/clasificación , Látex/análisis , Péptido Hidrolasas , Sapium/clasificación , Electroforesis en Gel de PoliacrilamidaRESUMEN
Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus fumigatus/aislamiento & purificación , Azotobacter/enzimología , Azotobacter/aislamiento & purificación , Fermentación , Metaloexopeptidasas/análisis , Metaloexopeptidasas/aislamiento & purificación , Péptido Hidrolasas/análisis , Serina/análisis , Activación Enzimática , Métodos , Estándares de Referencia , MétodosRESUMEN
We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.
Asunto(s)
Reactores Biológicos , Bacillus subtilis/enzimología , Bacillus subtilis/aislamiento & purificación , Fermentación , Péptido Hidrolasas/análisis , alfa-Amilasas/análisis , Activación Enzimática , Cinética , Métodos , Estándares de ReferenciaRESUMEN
A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.
Asunto(s)
Arthrodermataceae/enzimología , Arthrodermataceae/aislamiento & purificación , Secuencia de Bases , Microsporum/enzimología , Microsporum/aislamiento & purificación , Péptido Hidrolasas/análisis , Queratinas/análisis , Activación Enzimática , Métodos , VirulenciaRESUMEN
Various cultivation parameters were optimized for the production of extra cellular protease by Brevibacterium linens DSM 20158 grown in solid state fermentation conditions using statistical approach. The cultivation variables were screened by the Plackett-Burman design and four significant variables (soybean meal, wheat bran, (NH4)2SO4 and inoculum size were further optimized via central composite design (CCD) using a response surface methodological approach. Using the optimal factors (soybean meal 12.0g, wheat bran 8.50g, (NH4)2SO4) 0.45g and inoculum size 3.50%), the rate of protease production was found to be twofold higher in the optimized medium as compared to the unoptimized reference medium.
Asunto(s)
Brevibacterium/enzimología , Brevibacterium/aislamiento & purificación , Fermentación , Glycine max/enzimología , Péptido Hidrolasas/análisis , Condición del Suelo , Triticum/enzimología , Activación Enzimática , Harina , Métodos , Estándares de Referencia , Interpretación Estadística de DatosRESUMEN
Introducción. Las úlceras crónicas son una afección con un impacto negativo importante en la calidad de vida de los pacientes y en el sistema de salud; la aparición de infecciones y su difícil manejo, así como la presencia de tejido necrótico, afectan el pronóstico de curación. La larvaterapia se presenta como una opción para el desbridamiento y el manejo de infecciones de úlceras crónicas. Objetivo. Evaluar la larvaterapia en heridas con poca carga de tejido necrótico y evaluar las excreciones, secreciones y la hemolinfa de las larvas, respecto a su contenido enzimático. Materiales y métodos. Se reporta una serie de tres casos clínicos con úlceras crónicas y poca carga de tejido necrótico, tratados con larvaterapia, y se evalúa su evolución por los índices PUSH (Pressure Ulcer Scale for Healing) y Wound Bed Score, así como el patrón electroforético y contenido enzimático por zimograma de las excreciones y secreciones, y de la hemolinfa de las larvas. Resultados. Con solo una aplicación de la larvaterapia se evidenció una mejoría del aspecto de la herida y en los puntajes evaluados; en el PUSH hubo una disminución de 2,3 puntos, en promedio, y con el Wound Bed Score, un incremento de 2,7, lo que demuestra una mejoría en ambas escalas. Conclusión. Se encontró una actividad enzimática diversa en su contenido de excreciones y secreciones, con predominio de actividad de la proteasa de tipo serina.
Introduction. Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. Objective. The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. Results. The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demontrated with major activity belonging to serine protease family. Conclusions. Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.
Asunto(s)
Adulto , Anciano , Animales , Femenino , Humanos , Masculino , Terapia Biológica , Desbridamiento/métodos , Pie Diabético/terapia , Proteínas de Insectos/análisis , Larva/enzimología , Péptido Hidrolasas/análisis , Úlcera Varicosa/terapia , Antibacterianos/uso terapéutico , Terapia Combinada , Pie Diabético/patología , Dípteros/enzimología , Dípteros/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Hemolinfa/enzimología , Proteínas de Insectos , Necrosis , Manejo del Dolor , Péptido Hidrolasas , Índice de Severidad de la Enfermedad , Úlcera Varicosa/tratamiento farmacológico , Úlcera Varicosa/patología , Cicatrización de HeridasRESUMEN
Phytases are a group of enzymes that catalyze phytic acid hydrolysis with release of phosphorus (P). The ability of Chromobacterium sp. to produce phytase was detected in 115 out of 118 candidate bacteria isolated from different Brazilian biomas. This is the first report revealing the genus Chromobacterium as phytase producer.
Asunto(s)
Secuencia de Bases , Biomasa , Chromobacterium/enzimología , Chromobacterium/aislamiento & purificación , Microbiología Ambiental , Reactivadores Enzimáticos , Eutrofización , Monoéster Fosfórico Hidrolasas , Péptido Hidrolasas/análisis , Catálisis , Activación Enzimática , Variación Genética , Hidrólisis , Métodos , Métodos , Ecosistema TropicalRESUMEN
Growth and nitrilase production by recombinant Escherichia coli cells harbouring pET 21 (b) plasmid, for the expression of Pseudomonas putida nitrilase were improved using response surface methodology. Central composite design was used for obtaining ideal concentration of critical medium components which include fructose, tryptone, yeast extract and lactose. The optimal values for the concentration of fructose, tryptone, yeast extract and lactose were found to be 1.13, 2.26, 3.25 and 0.9 percent (w/v), respectively. Here, fructose served as carbon source for the growth while lactose was preferably used as inducer for the expression of foreign protein. Yeast extract in the medium was used as a growth promoter while tryptone was added as a major nitrogen source. Using this optimized medium, an experimental growth of 6.67 (OD at 600 nm) and nitrilase activity of 27.13 U/ml was achieved. This approach for medium development led to an enhancement of the growth and enzyme activity by 1.4 and 2.2 times, respectively, as compared to the un-optimized medium.
Asunto(s)
Aminohidrolasas/análisis , Nitrilos/análisis , Péptido Hidrolasas/análisis , Proteínas Recombinantes/análisis , Proteínas de Escherichia coli/análisis , Catálisis , Activación Enzimática , Métodos , MétodosRESUMEN
Litopenaeus vannamei, which is the most common shrimp species cultivated in the northeast of Brazil, is very susceptible to microbial diseases, and this consequently affects productivity. There are reports of bacteria, viruses and protozoa in these shrimp, but not fungi. This study aims to isolate and identify fungi present in shrimp Litopenaeus vannamei, and in their nursery waters, at two breeding farms in Brazil. The pathogenic potential of the isolates was assessed through the qualitative detection of proteases and aflatoxin B production. The 146 isolated fungi comprised 46 species. Aspergillus, Penicillium and Furarium were the three most relevant genera and Aspergillus flavus was the predominant species with a total of 33 isolates. Most of the isolated species are known as potentially pathogenic to humans and other animals. Eighteen isolates of A. flavus and two of A. parasiticus were able to produce aflatoxin B and 33 out of the 46 species produced protease, indicating that these fungi may also become pathogenic to shrimp and their consumers.
Asunto(s)
Aflatoxinas/análisis , Aflatoxinas/aislamiento & purificación , Biodiversidad , Hongos Mitospóricos/enzimología , Hongos Mitospóricos/aislamiento & purificación , Penaeidae/enzimología , Penaeidae/patogenicidad , Péptido Hidrolasas/análisis , Péptido Hidrolasas/aislamiento & purificación , Diagnóstico , Muestras de Alimentos , Métodos , Métodos , VirulenciaRESUMEN
Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 percent NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98 percent sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 percent activity at 80 0C after 4 h and more than 70 percent activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 percent active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.
Asunto(s)
Amilasas/genética , Amilasas/aislamiento & purificación , Catalasa/análisis , Catalasa/aislamiento & purificación , Leche/enzimología , Marinobacter/genética , Marinobacter/aislamiento & purificación , Péptido Hidrolasas/análisis , Análisis de Secuencia de ADN , Muestras de Alimentos , Microbiología Industrial , Métodos , MétodosRESUMEN
Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.