Producción de lipasas a partir de hongos endofíticos de Aniba canelilla, caracterización y aplicación del extracto enzimático

Endophytic fungi (EF) have a notable capacity to produce active molecules of industrial importance, such as hydrolytic enzymes. In this study we investigated the production of lipase by EFs isolated from the Amazonian species Aniba canelilla (Lauraceae), characterized the enzymatic extract obtained from the most promising fungus, and applied the lipolytic extract as a biocatalyst in the transesterification reaction for biodiesel production. The fungi were submitted to enzymatic screening in solid medium and in submerged fermentation to assess their lipase production. A total of 292 fungi were tested in solid media. Lipolytic activity was detected in 74% of the fungi cultivated in liquid media, 18 of which showing promising enzymatic production. The best lipase producer, Endomelanconiopsis endophytica QAT_7AC, was identified by sequencing of the ITS region. After adjusting the bioprocess conditions, E. endophytica QAT_7AC produced 2,415.5 U/mL of lipase after 72 h. The enzymatic extract showed higher lipolytic activity under pH 8.0 and 40 oC. The extract was applied as a biocatalyst in a transesterification reaction performed at 40 oC, with ethanol and waste cooking oil (3:1). The biodiesel yield was found to be 87% after 2 h rection when the fungal enzyme was used and 89% with the commercial biocatalyst. The endophytic fungi isolated from A. canelilla proved themselves to be biotechnologically relevant, as they can be explored as potential producers of lipases. The lipolytic extract can be applied in the synthesis of biodiesel using waste cooking oil.Os fungos endofíticos (FE) possuem notável capacidade de produzir moléculas ativas de importância industrial, como as enzimas hidrolíticas. Neste estudo foi investigada a produção de lipase por FEs isolados da espécie amazônica Aniba canelilla (Lauraceae), sendo caracterizado o extrato enzimático obtido a partir do fungo mais promissor e avaliada a aplicação do extrato lipolítico como biocatalisador na reação de transesterificação para a produção de biodiesel. Os fungos foram submetidos à triagem enzimática em meio sólido e por fermentação submersa, para verificar a produção de lipase. Um total de 292 fungos foram testados em meio sólido. A atividade lipolítica detectada em 74% dos fungos cultivados em meio líquido, onde 18 apresentaram atividade enzimática promissora. O melhor produtor de lipase, Endomelanconiopsis endophytica QAT_7AC, foi identificado pelo sequenciamento da região ITS. Após o ajuste das condições do bioprocesso, o E. endophytica QAT_7AC produziu 2.415,5 U/mL de lipase em 72 h. O extrato enzimático apresentou maior atividade lipolítica em pH 8,0 e 40 °C. O extrato foi aplicado como biocatalisador em uma reação de transesterificação realizada a 40 °C, com etanol e óleo de cozinha residual (3:1). O rendimento de biodiesel foi de 87% após 2 h de reação quando a enzima fúngica foi utilizada e de 89% com o biocatalisador comercial. Os fungos endofíticos isolados de A. canelilla mostraram-se biotecnologicamente relevantes e podem ser explorados como potenciais produtores de lipases. O extrato lipolítico pode ser aplicado na síntese de biodiesel a partir do óleo de cozinha residual.Los hongos endofíticos (EF) tienen una notable capacidad de producir moléculas activas de importancia industrial, como las enzimas hidrolíticas. En este estudio investigamos la producción de lipasa por hongos aislados de la especie Amazónica Aniba canelilla (Lauraceae), caracterizamos el extracto enzimático obtenido de lo hongo más promisor y aplicamos el extracto lipolítico como biocatalizador en la reacción de transesterificación para la producción de biodiésel. Los hongos aislados fueron sometidos a un cribado enzimático en medio sólido y fermentación sumergida para avaluar la producción de lipasa. Un total de 292 hongos fueron testados. La actividad lipolítica fue detectada en el 74% de los hongos cultivados en medio líquido, 18 de los cuales presentaron producción enzimática promisoria. El mejor productor de lipasa, Endomelanconiopsis endophytica QAT_7AC, fue identificado por el secuenciamento de la región ITS. Después del ajuste de las condiciones del bioproceso, E. endophytica QAT_7AC produjo 2,415.5 U/mL de lipasa en 72 h. El extracto enzimático presento mayor actividad lipolítica a pH 8.0 y 40 oC. El extracto enzimático fue aplicado como biocatalizador en una reacción de transesterificación realizada a 40 oC con etanol y aceite de cocina usado (3:1). El rendimiento del biodiesel fue de 87% después de 2 h de reacción cuando la enzima fúngica fue utilizada y 89% con el biocatalizador comercial. Los hongos endofíticos aislados de A. canelilla fueron biotecnológicamente relevantes, y pueden ser explorados como potenciales productores de lipasas. El extracto enzimático lipolítico puede ser aplicado en la síntesis de biodiesel utilizando aceite de cocina usado

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants

Metabolitos de Aspergillus fumigatus endofítico e seu efeito in vitro contra o agente causal da tuberculose

Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 μg mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.A tuberculose continua a ser uma das doenças infecciosas transmissíveis mais mortais, causando 1,4 milhão de mortes em 2015 em todo o mundo devido a vários fatores, incluindo o tratamento inadequado e o surgimento de cepas multirresistentes do agente causal, Mycobacterium tuberculosis. Portanto, as drogas desenvolvidas a partir de fontes naturais, como micro-organismos e extratos de plantas, são um alvo freqüente para a pesquisa e descoberta de compostos antimicrobianos. O presente estudo foi um ponto de partida para caracterizar compostos produzidos por um Aspergillus fumigatus isolado de copaíba (Copaifera multijuga) que inibe eficientemente M. tuberculosis, liberando os compostos no caldo de fermentação em condições de cultura específicas. Realizou-se um ensaio preliminar com uma espécie correlata, M. smegmatis, com o objetivo de detectar um efeito antimicrobiano relacionado ao caldo de fermentação de A. fumigatus. O uso direto deste substrato em ensaios de antibiose contra a estirpe H37Rv de M. tuberculosis (ATCC 27294) permitiu a detecção de atividade antimicrobiana com uma concentração inibitória mínima de 256 μg mL-1, demonstrando que os processos de purificação desenvolvidos pelo Biotage Flash Chromatography System são técnicas robustas e confiáveis para purificar compostos de fontes naturais. Além disso, este sistema cromatográfico pode ser usado em combinação com testes bioquímicos específicos, melhorando a busca de resultados confiáveis. Concluímos que esta fração pode expressar uma ampla gama de ação, inibindo ambas as espécies de Mycobacterium utilizadas como organismos-alvo

Polyhydroxyalkanoate (PHA) production by Lynisibacillus sp. strain UEA-20.171

PHA is a biodegradable and biocompatible natural thermoplastic produced from renewable bioresources and is hence attracting attention as a plastic material for use in the environment and medical fields. In the present study, the Lynisibacillus sp. strain UEA-20.171 was selected for production of polyhydroxyalkanoate (PHA) in bioreactor. The accumulation of polymer in the bacterium occurs when there is an imbalance in the concentration of nitrogen in relation to carbon (glucose). After the period of adaptation in mineral medium from 12 to 24 h, the cell division occurred from 12 to 24 h, and the formation of biopolymers from 36 to 48 h. It was observed that the polymer produced by the isolated UEA-20.171, was P(3HB).Keywords: Polyhydroxyalkanoates, polyhydroxybutyrate, bioplastics, Lysinibacillu

Kinetic Activity of Chromosomes and Expression of Recombination Genes in Achiasmatic Meiosis of Tityus (Archaeotityus) Scorpions

Several species of Tityus (Scorpiones, Buthidae) present multi-chromosomal meiotic associations and failures in the synaptic process, originated from reciprocal translocations. Holocentric chromosomes and achiasmatic meiosis in males are present in all members of this genus. In the present study, we investigated synapse dynamics, transcriptional silencing by γH2AX, and meiotic microtubule association in bivalents and a quadrivalent of the scorpion Tityus maranhensis. Additionally, we performed RT-PCR to verify the expression of mismatch repair enzymes involved in crossing-over formation in Tityus silvestris gonads. The quadrivalent association in T. maranhensis showed delay in the synaptic process and long asynaptic regions during pachytene. In this species, γH2AX was recorded only at the chromosome ends during early stages of prophase I; in metaphase I, bivalents and quadrivalents of T. maranhensis exhibited binding to microtubules along their entire length, while in metaphase II/anaphase II transition, spindle fibers interacted only with telomeric regions. Regarding T. silvestris, genes involved in the recombination process were transcribed in ovaries, testes and embryos, without significant difference between these tissues. The expression of these genes during T. silvestris achiasmatic meiosis is discussed in the present study. The absence of meiotic inactivation by γH2AX and holo/telokinetic behavior of the chromosomes are important factors for the maintenance of the quadrivalent in T. maranhensis and the normal continuation of the meiotic cycle in this species

Metabolites from endophytic Aspergillus fumigatus and their in vitro effect against the causal agent of tuberculosis

<div><p>ABSTRACT Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 μg mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.</p></div