New aspects on atrazine biodegradation

The world practice of using agrochemicals for long periods, in an indiscriminated and abusive way, has been a concern of the authorities involved in public health and sustainability of the natural resources, as a consequence of environmental contamination. Agrochemicals refer to a broad range of insecticides, fungicides and herbicides, and among them stands out atrazine, a herbicide intensively used in sugarcane, corn and sorghum cultures, among others. Researches have demonstrated that atrazine has toxic effects in algae, aquatic plants, aquatic insects, fishes and mammals. Due to the toxicity and persistence of atrazine in the environment, the search of microbial strains capable of degrading it is fundamental to the development of bioremediation processes, as corrective tools to solve the current problems of the irrational use of agrochemicals. This review relates the main microbial aspects and research on atrazine degradation by isolated microbial species and microbial consortia, as well as approaches on the development of techniques for microbial removal of atrazine in natural environments

Xylanase from Fusarium heterosporum: Properties and influence of thiol compounds on xylanase activity

The properties of xylanase purified from Fusarium heterosporum that was grown in barley-brewing residue under solid-state fermentation and the effects of thiol compounds on the reactivation of the metal ion-inhibited xylanase were investigated. Xylanase was purified to homogeneity by ion exchange chromatography, and its molecular mass was estimated to be 19.5 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the xylanase was 5.0, and it was stable in acidic pH (4.5 to 5.5), where it retained more than 87% of its activity after 24 h. The optimum temperature was 50°C, and it had a half-life of 53 min at 45°C. The apparent Km and Vmax values for the xylanase were 5.63 mg/ml and 800 μmol/mg/min, respectively. Ba2+, Ca2+, Mg2+ and the thiol compounds β-mercaptoethanol and dithiothreitol (DTT) enhanced xylanase activity, while Hg2+, Pb2+ and Zn2+ strongly inhibited enzyme activity. Furthermore, this xylanase had an alternative mode of regulation in the presence of thiol compounds because the enzyme was able to recover its catalytic activity after inhibition by heavy metal ions.Keywords: Hemicellulase, fungus, solid-state fermentation, barley brewing residue, thiol compoundsAfrican Journal of Biotechnology, Vol. 13(9), pp. 1047-1055, 26 February, 201

Efeito da dose resposta de Paracoccidioides brasiliensis no modelo experimental de artrite

A paracoccidioidomicose (PCM) é causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb) e corresponde à micose sistêmica de maior prevalência na América Latina. O objetivo do presente trabalho foi avaliar a dose resposta de leveduras do fungo para padronização do modelo experimental de artrite séptica. Os experimentos foram realizados com grupos de 14 ratos que receberam doses de 103, 104 ou 105 células de P. brasiliensis (Pb18). Os fungos foram injetados em 50 µL de solução salina em tampão fosfatado (PBS) diretamente na articulação do joelho dos animais. Os seguintes parâmetros foram analisados neste trabalho: a formação de edema nos joelhos infundidos com as células das leveduras e alterações radiológicas, anatopalógicas além de titulação de anticorpos por Elisa. Após 15 dias de infecção, os sinais de inflamação foram evidentes. Aos 45 dias, algumas características de dano e necrose foram observadas na cartilagem articular. A disseminação sistêmica do fungo foi observada em 11% dos animais inoculados, concluiu-se que o modelo experimental é capaz de mimetizar a PCM articular em humanos e que a dose de 105 leveduras representa a dose padrão para o desenvolvimento do modelo.Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model

A thermostable xylanase from a new strain of Aspergillus fumigatus presents high ability to hydrolyze hemicellulose from corn straw / Uma xilanase termoestável de uma nova estirpe de Aspergillus fumigatus apresenta elevada capacidade de hidrolisar hemicelulose a partir de palha de milho

ABSTRACTIn order to optimize the production of xylanase from a new thermophilic strain of Aspergillus fumigatus (OI-1R-T), Plackett-Burman design (PBD) and central composite rotational design (CCRD) were performed. The response surface plots indicated a trend for increased xylanase biosynthesis with increasing concentrations of corn straw. The optimized xylanase activity was 530 U mL-1 in the presence of 6.5% (w/v) of the residual biomass, which was 11 times (1,157%) higher than that obtained with only the PBD (45.8 U mL-1). Interestingly, xylanase thermostability was maintained at 90% at 50 °C for 6 h. Enzymatic hydrolysis assays conducted for 96 h with 2 U mL-1 of xylanase and crude corn straw, pre-treated corn straw (hemicellulose) and xylan from beechwood, resulted in the net production of 3.89, 20.96 and 21.64 µmol mL-1of reducing sugars, respectively. Thus, A. fumigatus xylanase was equally able to hydrolyzes hemicellulose from corn straw and xylan from beechwood. The present data indicate that the xylanase activity of A. fumigatus could be applied to the production of low molecular weight sugars for use by pentose-fermenting yeast for the production of fuels and chemicals, among other products. 

Structure, expression and functional analysis of the gene encoding Calmodulin in the aquatic fungus Blastocladiella emersonii

O único gene para Calmodulina (CaM) e o cDNA correspondente do quitridiomiceto Blastocladiella emersonii foram isolados e caracterizados. O gene CaM é interrompido por três introns e transcrito como uma única espécie de mRNA de 0,7 kb, codificando uma proteína 91% idêntica à CaM humana. A Calmodulina de B. emersonii foi expressa em Escherichia coli como uma proteína de fusão com Glutationa-S-transferase (GST), purificada por cromatografia de afinidade e clivada da porção GST usando uma protease sítio específico. Na presença de Ca2+, a CaM de B. emersonii exibiu uma alteração na mobilidade eletroforética semelhante à CaM bovina e foi capaz de ativar a autofosforilação da proteína quinase dependente de Ca+2-CaM (CaMKII) de cérebro de rato. A expressão da Calmodulina é regulada durante o desenvolvimento em B. emersonii, o mRNA da CaM e a concentração da proteína aumentam durante a esporulação atingindo um nível máximo antes da liberação dos zoósporos no meio, no final deste estágio. Os níveis da proteína e do mRNA decrescem drasticamente no estágio de zoósporo aumentando novamente durante a germinação. Os antagonistas de CaM, composto 48/80, calmidazolium e W7 inibiram completamente a esporulação de B. emersonii quando adicionados às culturas pelo menos 120, 150 e 180 min após a indução, respectivamente. Todas estas drogas também inibiram o crescimento e a produção de zoósporos neste fungo. O bloqueador de canais de Ca+2 , TMB-8, e o inibidor de CaMKII KN93 inibiram completamente a esporulação quando adicionados até 60 minutos após a indução deste estágio, porém apenas KN93 afetou o crescimento do fungo. Os dados apresentados sugerem que o complexo Ca+2-CaM e a CaMKII têm um papel importante durante o crescimento e esporulação em B. emersonii.The single Calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0,7 kb mRNA species, encoding a predicted protein 91% identical to the human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with Gluthatione-S-transferase (GST), and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca2+, B. emersonii CaM exhibited a shift in apparent Mr similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, CaM mRNA and protein concentrations increasing during sporulation with maximum levels observed prior to the release of the zoospores to the medium at the end of this stage. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists, compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150 and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca2+ channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that Ca2+-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii

Structure, expression and functional analysis of the gene encoding Calmodulin in the aquatic fungus Blastocladiella emersonii

O único gene para Calmodulina (CaM) e o cDNA correspondente do quitridiomiceto Blastocladiella emersonii foram isolados e caracterizados. O gene CaM é interrompido por três introns e transcrito como uma única espécie de mRNA de 0,7 kb, codificando uma proteína 91% idêntica à CaM humana. A Calmodulina de B. emersonii foi expressa em Escherichia coli como uma proteína de fusão com Glutationa-S-transferase (GST), purificada por cromatografia de afinidade e clivada da porção GST usando uma protease sítio específico. Na presença de Ca2+, a CaM de B. emersonii exibiu uma alteração na mobilidade eletroforética semelhante à CaM bovina e foi capaz de ativar a autofosforilação da proteína quinase dependente de Ca+2-CaM (CaMKII) de cérebro de rato. A expressão da Calmodulina é regulada durante o desenvolvimento em B. emersonii, o mRNA da CaM e a concentração da proteína aumentam durante a esporulação atingindo um nível máximo antes da liberação dos zoósporos no meio, no final deste estágio. Os níveis da proteína e do mRNA decrescem drasticamente no estágio de zoósporo aumentando novamente durante a germinação. Os antagonistas de CaM, composto 48/80, calmidazolium e W7 inibiram completamente a esporulação de B. emersonii quando adicionados às culturas pelo menos 120, 150 e 180 min após a indução, respectivamente. Todas estas drogas também inibiram o crescimento e a produção de zoósporos neste fungo. O bloqueador de canais de Ca+2 , TMB-8, e o inibidor de CaMKII KN93 inibiram completamente a esporulação quando adicionados até 60 minutos após a indução deste estágio, porém apenas KN93 afetou o crescimento do fungo. Os dados apresentados sugerem que o complexo Ca+2-CaM e a CaMKII têm um papel importante durante o crescimento e esporulação em B. emersonii.The single Calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0,7 kb mRNA species, encoding a predicted protein 91% identical to the human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with Gluthatione-S-transferase (GST), and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca2+, B. emersonii CaM exhibited a shift in apparent Mr similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, CaM mRNA and protein concentrations increasing during sporulation with maximum levels observed prior to the release of the zoospores to the medium at the end of this stage. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists, compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150 and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca2+ channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that Ca2+-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii

New aspects on atrazine biodegradation

The world practice of using agrochemicals for long periods, in an indiscriminated and abusive way, has been a concern of the authorities involved in public health and sustainability of the natural resources, as a consequence of environmental contamination. Agrochemicals refer to a broad range of insecticides, fungicides and herbicides, and among them stands out atrazine, a herbicide intensively used in sugarcane, corn and sorghum cultures, among others. Researches have demonstrated that atrazine has toxic effects in algae, aquatic plants, aquatic insects, fishes and mammals. Due to the toxicity and persistence of atrazine in the environment, the search of microbial strains capable of degrading it is fundamental to the development of bioremediation processes, as corrective tools to solve the current problems of the irrational use of agrochemicals. This review relates the main microbial aspects and research on atrazine degradation by isolated microbial species and microbial consortia, as well as approaches on the development of techniques for microbial removal of atrazine in natural environments