A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production

<p>Abstract</p> <p>Background</p> <p>Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. <it>Actinobacillus succinogenes </it>naturally converts sugars and CO₂into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process.</p> <p>Results</p> <p>Described here is the 2.3 Mb <it>A. succinogenes </it>genome sequence with emphasis on <it>A. succinogenes</it>'s potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that <it>A. succinogenes </it>is capable of natural transformation. <it>A. succinogenes </it>lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of <it>A. succinogenes </it>and its closest known relative, <it>Mannheimia succiniciproducens</it>, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of <it>A. succinogenes </it>genomic features in other sequenced Pasteurellaceae.</p> <p>Conclusions</p> <p>Both <it>A. succinogenes </it>and <it>M. succiniciproducens </it>genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the <it>A. succinogenes </it>genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, <it>A. succinogenes</it>'s natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by <it>A. succinogenes</it>.</p

Comparative analysis of thermophilic and mesophilic proteins using Protein Energy Networks

<p>Abstract</p> <p>Background</p> <p>Thermophilic proteins sustain themselves and function at higher temperatures. Despite their structural and functional similarities with their mesophilic homologues, they show enhanced stability. Various comparative studies at genomic, protein sequence and structure levels, and experimental works highlight the different factors and dominant interacting forces contributing to this increased stability.</p> <p>Methods</p> <p>In this comparative structure based study, we have used interaction energies between amino acids, to generate structure networks called as Protein Energy Networks (PENs). These PENs are used to compute network, sub-graph, and node specific parameters. These parameters are then compared between the thermophile-mesophile homologues.</p> <p>Results</p> <p>The results show an increased number of clusters and low energy cliques in thermophiles as the main contributing factors for their enhanced stability. Further more, we see an increase in the number of hubs in thermophiles. We also observe no community of electrostatic cliques forming in PENs.</p> <p>Conclusion</p> <p>In this study we were able to take an energy based network approach, to identify the factors responsible for enhanced stability of thermophiles, by comparative analysis. We were able to point out that the sub-graph parameters are the prominent contributing factors. The thermophiles have a better-packed hydrophobic core. We have also discussed how thermophiles, although increasing stability through higher connectivity retains conformational flexibility, from a cliques and communities perspective.</p

Damage of woven composite under tensile and shear stress using infrared thermography and micrographic cuts

Infrared thermography was used to study damage developing in woven fabrics. Two different experiments were performed, a ±45° tensile test and a rail shear test. These two different types of tests show different damage scenarios, even if the shear stress/strain curves are similar. The ±45° tension test shows matrix hardening and matrix cracking whereas the rail shear test shows only matrix hardening. The infrared thermography was used to perform an energy balance, which enabled the visualization of the portion of dissipated energy caused by matrix cracking. The results showed that when the resin is subjected to pure shear, a larger amount of energy is stored by the material, whereas when the resin is subjected to hydrostatic pressure, the main part of mechanical energy is dissipated as heat

Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation

An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70 °C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60 °C and up to 97 % in the pH range 7.5–10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K+ that showed a stimulating effect, and Fe2+, Co2+ and Hg2+, which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25 °C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation

Environmental factors modulating the stability and enzymatic activity of the Petrotoga mobilis Esterase (PmEst)

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders

Stability mechanisms of a thermophilic laccase probed by molecular dynamics.

Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ∼0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (∼9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching T opt (∼350 K) from 300 K, this change correlated with a beginning "unzipping" of critical β-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F(-) (but not Cl(-)) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ∼4 across simulations, mainly via prevention of F(-) intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical β-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes