Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100

Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROS-induced nitrogenase inhibition in Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus, an endophyte isolated from sugarcane, is a strict aerobe that fixates N2. This process is catalyzed by nitrogenase and requires copious amounts of ATP. Nitrogenase activity is extremely sensitive to inhibition by oxygen and reactive oxygen species (ROS). However, the elevated oxidative metabolic rates required to sustain biological nitrogen fixation (BNF) may favor an increased production of ROS. Here, we explored this paradox and observed that ROS levels are, in fact, decreased in nitrogen-fixing cells due to the up-regulation of transcript levels of six ROS-detoxifying genes. A cluster analyses based on common expression patterns revealed the existence of a stable cluster with 99.8% similarity made up of the genes encoding the α-subunit of nitrogenase Mo–Fe protein (nifD), superoxide dismutase (sodA) and catalase type E (katE). Finally, nitrogenase activity was inhibited in a dose-dependent manner by paraquat, a redox cycler that increases cellular ROS levels. Our data revealed that ROS can strongly inhibit nitrogenase activity, and G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels resulting in a lower ROS generation. We suggest that careful controlled ROS production during this critical phase is an adaptive mechanism to allow nitrogen fixation

Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation

8 p. : il.Background and aims Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium isolated from sugarcane, rice, elephant grass, sweet potato, coffee, and pineapple. These plants have high level of asparagine, which promotes microbial growth and inhibits nitrogenase activity. The regulation of intracellular concentrations of this amino acid is essential for growth and biological nitrogen fixation (BNF) in this diazotroph; however its asparagine metabolic pathway has not yet been clearly established. Methods The work reported here is the first to demonstrate the use of an alternative route for asparaginyl-tRNA (Asn-tRNA) and asparagine formation in an endophytic nitrogen-fixing bacterium by using in silico and in vitro analysis. Results The indirect route involves transamidation of incorrectly charged tRNA via GatCAB transamidase. Nitrogenase activity was completely inhibited by 20 mM Asn in LGI-P medium, which in contrast promotes protein synthesis and microbial growth. Conclusions The analysis carried out in this work shows that intracellular levels of asparagine regulate the expression of nitrogenase nifD gene (GDI0437), suggesting that the presence of an alternative route to produce asparagine might give the G. diazotrophicus a tighter control over cell growth and BNF, and may be of importance in the regulation of the endophytic plant-microbe interaction

Explorando as aplicações biotecnológicas do domínio Archaea

Submitted by Alexandre Sousa ([email protected]) on 2015-12-16T10:28:15Z No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-12-16T10:39:55Z (GMT) No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5)Made available in DSpace on 2015-12-16T10:39:55Z (GMT). No. of bitstreams: 1 Braz_J_Microbiol_38_398-405.pdf: 488726 bytes, checksum: b81824bef49afc9dfa9158af9528d14d (MD5) Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Química. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.As arqueas representam uma considerável fração dos procariotos nos ecossistemas marinhos e terrestes, indicando que estes organismos devem possuir um grande impacto nos ciclos energéticos. A natureza extremofílica de muitas arqueas tem estimulado intensos esforços para compreender sua adaptação fisiológica a ambientes extremos. Suas propriedades incomus as tornam uma fonte valiosa no desenvolvimento de novos processos biotecnológicos e aplicações industriais como novos fármacos, cosméticos, suplementos nutricionais, sondas moleculares, enzimas e reagentes. Na presente mini-revisão, mostramos e discutimos algumas de suas características exclusivas correlacionando-as com seu potencial biotecnológico e aplicação industrial. Os tópicos são: características das arqueas, divisão filogenética, aplicações biotecnológicas, isolamento e cultivo de novos microrganismos, genoma e metagenoma.Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. The extremophilic nature of many archaea has stimulated intense efforts to understand the physiological adaptations for living in extreme environments. Their unusual properties make them a potentially valuable resource in the development of novel biotechnological processes and industrial applications as new pharmaceuticals, cosmetics, nutritional supplements, molecular probes, enzymes, and fine chemicals. In the present mini-review, we show and discuss some exclusive characteristics of Archaea domain and the current knowledge about the biotechnological uses of the archaeal enzymes. The topics are: archaeal characteristics, phylogenetic division, biotechnological applications, isolation and cultivation of new microbes, achievements in genomics, and metagenomic

Archaea: potencial biotecnológico

Submitted by Alexandre Sousa ([email protected]) on 2015-11-09T16:49:10Z No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2015-11-09T17:06:54Z (GMT) No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5)Made available in DSpace on 2015-11-09T17:06:54Z (GMT). No. of bitstreams: 1 Biotec Cienc Desenv_30.pdf: 256940 bytes, checksum: 79b715b41243787328bc6b1c614a3e4a (MD5) Previous issue date: 2003Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Biologia. Departamento de Biologia Marinha. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, COPPE, Programa de Engenharia Química. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas. Departamento de Bioquímica Médica. Rio de Janeiro, RJ, Brasil

Complete genome sequence of the sugarcane nitrogen-fixing endophyte gluconacetobacter diazotrophicus PAL5

17 p. : il.Background: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. Results: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. Conclusion: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications