6 research outputs found
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 (TRXâPF2001Î60) and without the thioredoxin tag (PF2001Î60) 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 TRXâPF2001Î60 presented specific activity twice lower than the enzyme PF2001Î60. The enzyme PF2001Î60 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 PF2001Î60 was enhanced with Triton X-100
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 aplicaçÔes biotecnolĂłgicas do domĂnio Archaea
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Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de QuĂmica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em SaĂșde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.As arqueas representam uma considerĂĄvel fração dos procariotos nos ecossistemas marinhos e terrestes, indicando que estes organismos devem possuir um grande impacto nos ciclos energĂ©ticos. A natureza extremofĂlica de muitas arqueas tem estimulado intensos esforços para compreender sua adaptação fisiolĂłgica a ambientes extremos. Suas propriedades incomus as tornam uma fonte valiosa no desenvolvimento de novos processos biotecnolĂłgicos e aplicaçÔes industriais como novos fĂĄrmacos, cosmĂ©ticos, suplementos nutricionais, sondas moleculares, enzimas e reagentes. Na presente mini-revisĂŁo, mostramos e discutimos algumas de suas caracterĂsticas exclusivas correlacionando-as com seu potencial biotecnolĂłgico e aplicação industrial. Os tĂłpicos sĂŁo: caracterĂsticas das arqueas, divisĂŁo filogenĂ©tica, aplicaçÔes biotecnolĂłgicas, 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 biotecnolĂłgico
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Previous issue date: 2003Universidade Federal do Rio de Janeiro, Instituto de CiĂȘncias BiomĂ©dicas. Departamento de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em SaĂșde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de CiĂȘncias BiomĂ©dicas. Departamento de BioquĂmica MĂ©dica. 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 QuĂmica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de CiĂȘncias BiomĂ©dicas. Departamento de BioquĂmica MĂ©dica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro, Instituto de CiĂȘncias BiomĂ©dicas. Departamento de BioquĂmica MĂ©dica. 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