RESUMO
Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-L-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit® CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher KM value and a 22-times decrease of the kcat/KM ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (rs/rh) when α-L-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit® CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-L-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit® CM support without affecting the synthesis of fucosylated oligosaccharides.
Assuntos
Thermotoga maritima , alfa-L-Fucosidase , Fucose , Oligossacarídeos , Especificidade por Substrato , Thermotoga , Thermotoga maritima/metabolismo , alfa-L-Fucosidase/metabolismoRESUMO
To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.
Assuntos
Reatores Biológicos/normas , Fermentação , Hidrogênio/metabolismo , Consórcios Microbianos/genética , RNA Ribossômico 16S/genética , Anaerobiose , Clostridium/classificação , Clostridium/genética , Clostridium/metabolismo , Enterobacter/classificação , Enterobacter/genética , Enterobacter/metabolismo , Firmicutes/classificação , Firmicutes/genética , Firmicutes/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Lactobacillus/classificação , Lactobacillus/genética , Lactobacillus/metabolismo , América Latina , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/metabolismo , Thermotoga maritima/classificação , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Veillonellaceae/classificação , Veillonellaceae/genética , Veillonellaceae/metabolismoRESUMO
The clusters of regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system consists of an intriguing machinery of proteins that confer bacteria and archaea with immunity against phages and plasmids via an RNA-guided interference mechanism. Here, the cloning, recombinant expression in Escherichia coli BL21 (DE3), purification, crystallization and preliminary X-ray diffraction analysis of Csm2 from Thermotoga maritima are reported. Csm2 is thought to be a component of an important protein complex of the type IIIA CRISPR-Cas system, which is involved in the CRISPR-Cas RNA-guided interference pathway. The structure of Csm2 was solved via cadmium single-wavelength anomalous diffraction (Cd-SAD) phasing. Owing to its involvement in the CRISPR-Cas system, the crystal structure of this protein could be of importance in elucidating the mechanism of type IIIA CRISPR-Cas systems in bacteria and archaea.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Thermotoga maritima/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas Associadas a CRISPR/isolamento & purificação , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Eletricidade EstáticaRESUMO
We present a method for calculating the configurational-dependent diffusion coefficient of a globular protein as a function of the global folding process. Using a coarse-grained structure-based model, we determined the diffusion coefficient, in reaction coordinate space, as a function of the fraction of native contacts formed Q for the cold shock protein (TmCSP). We find nonmonotonic behavior for the diffusion coefficient, with high values for the folded and unfolded ensembles and a lower range of values in the transition state ensemble. We also characterized the folding landscape associated with an energetically frustrated variant of the model. We find that a low-level of frustration can actually stabilize the native ensemble and increase the associated diffusion coefficient. These findings can be understood from a mechanistic standpoint, in that the transition state ensemble has a more homogeneous structural content when frustration is present. Additionally, these findings are consistent with earlier calculations based on lattice models of protein folding and more recent single-molecule fluorescence measurements.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dobramento de Proteína , Thermotoga maritima/metabolismo , Aminoácidos , Simulação por Computador , Difusão , Entropia , Modelos Moleculares , TemperaturaRESUMO
A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 degrees C, pH 6.6, 3% NaCl and 0.15% MgCl2.6H2O. The DNA G+C content was 36.1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97.6 to 98.8%. However DNA-DNA relatedness values between these species and strain MET12T were lower than 70%. As strain MET12T (=DSM 14811T=CIP 107371T) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.