BrEPS: a flexible and automatic protocol to compute enzyme-specific sequence profiles for functional annotation

<p>Abstract</p> <p>Background</p> <p>Models for the simulation of metabolic networks require the accurate prediction of enzyme function. Based on a genomic sequence, enzymatic functions of gene products are today mainly predicted by sequence database searching and operon analysis. Other methods can support these techniques: We have developed an automatic method "BrEPS" that creates highly specific sequence patterns for the functional annotation of enzymes.</p> <p>Results</p> <p>The enzymes in the UniprotKB are identified and their sequences compared against each other with BLAST. The enzymes are then clustered into a number of trees, where each tree node is associated with a set of EC-numbers. The enzyme sequences in the tree nodes are aligned with ClustalW. The conserved columns of the resulting multiple alignments are used to construct sequence patterns. In the last step, we verify the quality of the patterns by computing their specificity. Patterns with low specificity are omitted and recomputed further down in the tree. The final high-quality patterns can be used for functional annotation. We ran our protocol on a recent Swiss-Prot release and show statistics, as well as a comparison to PRIAM, a probabilistic method that is also specialized on the functional annotation of enzymes. We determine the amount of true positive annotations for five common microorganisms with data from BRENDA and AMENDA serving as standard of truth. BrEPS is almost on par with PRIAM, a fact which we discuss in the context of five manually investigated cases.</p> <p>Conclusions</p> <p>Our protocol computes highly specific sequence patterns that can be used to support the functional annotation of enzymes. The main advantages of our method are that it is automatic and unsupervised, and quite fast once the patterns are evaluated. The results show that BrEPS can be a valuable addition to the reconstruction of metabolic networks.</p

Sustainability of bioenergy – Mapping the risks &amp; benefits to inform future bioenergy systems

Bioenergy is widely included in energy strategies for its GHG mitigation potential. Bioenergy technologies will likely have to be deployed at scale to meet decarbonisation targets, and consequently biomass will have to be increasingly grown/mobilised. Sustainability risks associated with bioenergy may intensify with increasing deployment and where feedstocks are sourced through international trade. This research applies the Bioeconomy Sustainability Indicator Model (BSIM) to map and analyse the performance of bioenergy across 126 sustainability issues, evaluating 16 bioenergy case studies that reflect the breadth of biomass resources, technologies, energy vectors and bio-products. The research finds common trends in sustainability performance across projects that can inform bioenergy policy and decision making. Potential sustainability benefits are identified for People (jobs, skills, income, energy access); for Development (economy, energy, land utilisation); for Natural Systems (soil, heavy metals), and; for Climate Change (emissions, fuels). Also, consistent trends of sustainability risks where focus is required to ensure the viability of bioenergy projects, including for infrastructure, feedstock mobilisation, techno-economics and carbon stocks. Emission mitigation may be a primary objective for bioenergy, this research finds bioenergy projects can provide potential benefits far beyond emissions - there is an argument for supporting projects based on the ecosystem services and/or economic stimulation they may deliver. Also given the broad dynamics and characteristics of bioenergy projects, a rigid approach of assessing sustainability may be incompatible. Awarding ‘credit’ across a broader range of sustainability indicators in addition to requiring minimum performances in key areas, may be more effective at ensuring bioenergy sustainability

Electricity and disinfectant production from wastewater: Microbial Fuel Cell as a self-powered electrolyser

This study presents a simple and sustainable Microbial Fuel Cell as a standalone, self-powered reactor for in situ wastewater electrolysis, recovering nitrogen from wastewater. A process is proposed whereby the MFC electrical performance drives the electrolysis of wastewater towards the self-generation of catholyte within the same reactor. The MFCs were designed to harvest the generated catholyte in the internal chamber, which showed that liquid production rates are largely proportional to electrical current generation. The catholyte demonstrated bactericidal properties, compared to the control (open-circuit) diffusate, and reduced observable biofilm formation on the cathode electrode. Killing effects were confirmed using bacterial kill curves constructed by exposing a bioluminescent Escherichia coli target, as a surrogate coliform, to catholyte where a rapid kill rate was observed. Therefore, MFCs could serve as a water recovery system, a disinfectant/cleaner generator that limits undesired biofilm formation and as a washing agent in waterless urinals to improve sanitation. This simple and ready to implement MFC system can convert organic waste directly into electricity and self-driven nitrogen along with water recovery. This could lead to the development of energy positive bioprocesses for sustainable wastewater treatment

Both reversible self-association and structural changes underpin molecular viscoelasticity of mAb solutions

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of “mAb1.” These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein–protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate