Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “termination engineering”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295 T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved p CO 2 , and requires exogenous vitamin B 12 . In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (~5.6 µM) O 2 concentrations. The abundance of a potentially more efficient CO 2 -fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb 3 -type terminal oxidase increased under O 2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O 2 -sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O 2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Thermal-fluctuation effects on small-scale statistics in turbulent gas flow

Kolmogorov's theory of turbulence assumes that the small-scale turbulent structures in the energy cascade are universal and are determined by the energy dissipation rate and the kinematic viscosity alone. However, thermal fluctuations, absent from the continuum description, terminate the energy cascade near the Kolmogorov length scale. Here, we propose a simple superposition model to account for the effects of thermal fluctuations on small-scale turbulence statistics. Furthermore, for compressible Taylor–Green vortex flow, we demonstrate that the superposition model in conjunction with data from direct numerical simulation of the Navier–Stokes equations yields spectra and structure functions that agree with the corresponding quantities computed from the direct simulation Monte Carlo method of molecular gas dynamics, verifying the importance of thermal fluctuations in the dissipation range.

42 ENGINEERING↗
DOE OSTI2020

Busbar Design and Optimization for Voltage Overshoot Mitigation of a Silicon Carbide High-Power Three-Phase T-Type Inverter

The silicon carbide (SiC) devices have faster switching speed than that of the conventional silicon (Si) devices, which however may cause excessive device voltage overshoot. Larger gate resistance can help to restrain the overshoot, it however slows down the switching speed and increases switching losses. There are other methods that can mitigate the voltage overshoot, e.g., using low-inductance busbars, adding snubber circuits, etc. In this article, the busbar design for a 250-kW SiC three-level T-type inverter is investigated. The current commutation loops (CCLs) are first analyzed using a single-phase equivalent circuit. Then the detailed busbar design methods, especially a 3-D busbar design concept, are proposed to select the optimal stacking order for the multilayer laminated busbar and to address constraints posed by the physical terminal arrangements of SiC modules and dc-link capacitors. The stray inductance in each CCL is extracted via a finite element analysis and validated on the actual inverter busbar prototypes using an impedance analyzer. To further minimize the busbar stray inductance, a hybrid busbar structure with printed circuit board based buffer circuit using high-frequency decoupling capacitors is designed and evaluated in this article. Lastly, the effectiveness of the designed busbars as well as the buffer circuit are validated using experimental studies.

42 ENGINEERING↗
DOE OSTI2023

1 kV GaN-on-Si Quasi-Vertical Schottky Rectifier

Here, this work demonstrates quasi-vertical GaN Schottky barrier diodes (SBDs) on 6-inch Si substrate with a breakdown voltage (BV) over 1 kV, the highest BV reported in vertical GaN-on-Si SBDs to date. The deep mesa inherently in quasi-vertical devices is leveraged to form a self-aligned edge termination, and the mesa sidewall is covered by the p-type nickel oxide (NiO) as a reduced surface field (RESURF) structure. This novel termination enables a parallel-plane junction electric field of 2.8 MV/cm. The device also shows low turn-on voltage of 0.5 V, and low specific on-resistance of 1.1 mΩ∙ cm 2 . Moreover, the device exhibits excellent overvoltage robustness under the continuous 800 V stress in the unclamped inductive switching test. These results show the good promise of the low-cost vertical GaN-on-Si power diodes.

42 ENGINEERING↗
DOE OSTI2020

High voltage MOSFET devices and methods of making the devices

A SiC MOSFET device having low specific on resistance is described. The device has N+, P-well and JFET regions extended in one direction (Y-direction) and P+ and source contacts extended in an orthogonal direction (X-direction). The polysilicon gate of the device covers the JFET region and is terminated over the P-well region to minimize electric field at the polysilicon gate edge. In use, current flows vertically from the drain contact at the bottom of the structure into the JFET region and then laterally in the X direction through the accumulation region and through the MOSFET channels into the adjacent N+ region. The current flowing out of the channel then flows along the N+ region in the Y-direction and is collected by the source contacts and the final metal. Methods of making the device are also described.

42 ENGINEERING↗
DOE OSTI2025

Sequential Infiltration Synthesis of Cadmium Sulfide Discrete Atom Clusters

Abstract Exposure of soft material templates to alternating volatile chemical precursors can produce inorganic deposition within the permeable template (e.g. a polymer thin film) in a process akin to atomic layer deposition (ALD). While such sequential infiltration synthesis (SIS) processes have now been demonstrated for many metal oxides, we report an SIS process for a transition metal sulfide – CdS. Gas phase dimethyl cadmium and hydrogen sulfide precursors infiltrated into poly(4‐vinylpyridine) thin films result in the 3D‐nucleation of clusters consistent with a cubane‐type Cd 4 S 4 core that are variably terminated with methyl, thiol and hydroxy capping ligands. First principles models and simulation of few‐atom Cd‐based clusters are consistent with electronic and vibrational spectroscopy and grazing‐incidence total X‐ray scattering measurements of 3D‐cluster‐arrays synthesized at 80 °C. The direct synthesis of few‐atom transition metal sulfide clusters within polymer thin films will provide a versatile new route to precision architectures for light‐absorbing materials including solar energy harvesting and conversion applications.

Jayaweera, Nuwanthaka P. [Material Science Divisio↗
DOE OSTI2020

Process-based balance of system cost modeling for offshore wind power plants in the United States

This paper describes the development of a process-based and open-source balance of system cost model that provides the capability to evaluate both existing and novel offshore wind technologies. Individual design and installation steps are represented with bottom-up engineering models that compute times and costs associated with the process; furthermore, operational constraints are assigned to each process so that delays caused by weather and presence of marine mammals may be accounted for in the overall project timeline. The model structure, assumptions, inputs, and results are vetted with industry partners and compared against actual projects for validation. Installation times show reasonable agreement with real data. Project cost sensitivities are investigated to compute the system-level impact of different design choices. First, individual vessel efficiencies are computed for varying numbers of installation vessels and weather time series to show the diminishing returns of more than two feeder barges. Then, array cable capital costs and installation times are determined for a representative project with different turbine sizes. These values quantify the cost-benefit tradeoffs and show a net-cost savings of decreasing numbers of turbines, increased turbine spacing, and fewer turbine terminations. These results demonstrate that the balance of system model features the accuracy, functionality, and accessibility to serve as the foundation for a wide range of analyses to identify cost reduction potentials for offshore wind energy in the United States.

17 WIND ENERGY↗
DOE OSTI2019

Ultra Efficient Light Duty Powertrain with Gasoline Low Temperature Combustion (Final Report)

This document details the work completed during Project DE-EE0006839 ‘Ultra Efficient Light Duty Powertrain with Gasoline Low Temperature Combustion’. All tasks from the project’s Statement of Project Objectives are discussed. Final demonstration data is also presented and discussed. Further project details can be found in the Quarterly Technicial Reports that were submitted during the course of the project and are stored on the EERE Project Management Center Website. The goal of this project was to develop, implement and demonstrate a low temperature combustion approach called Gasoline Direct Injection Compression Ignition (GDCI) that provides high thermal efficiency with low NOx and PM emissions. The project target was to demonstrate a 35% fuel economy improvement over the baseline vehicle while meeting Tier 3 emissions levels. At the start of this project, GDCI was a moderately mature combustion technology, due in large part to advances made during a Delphi Technologies program funded through an earlier Department of Energy Advanced Technology Powertrains (ATP) opportunity; under that earlier contract, DE-EE0003258, Delphi Technologies assembled a team with complementary industrial and academic experience, and led this team to advance the technology from the original fundamental work to the state of art embodied by a first-generation (Gen 1) multi-cylinder GDCI engine. Key activities during that project centered on engine and combustion chamber design, and development of fuel injection equipment, valvetrain and advanced controls. Performance results with the Gen 1 engine in a development vehicle demonstrated that GDCI had the capability to operate using Low Temperature Combustion (LTC) over a broad speed-load range. Based on benchmarking work, efficiency for the Gen 1 GDCI engine compared favorably overall to production light-duty diesel engines. The second DOE funded project, DE-EE0006839, focused on a number of technical risks and issues that were left to be resolved for GDCI to become a production-viable technology. These risks were:GDCI combustion system refinement to achieve near-ideal air/fuel mixture preparation for high efficiency and low HC and CO emissions; Transient control development for low temperature combustion with high EGR levels during real-world transient driving maneuvers and over a broader range of ambient conditions; Aftertreatment system design that is effective in dealing with the low-temperature challenges of a highly efficient engine. The project was executed in two phases. Phase 1 leveraged the development vehicle from the Advanced Technologies Powertrain 1 (ATP1) project, DE-EE0003258. That vehicle was used as an initial test bed in the development of a high performance low temperature exhaust aftertreatment system. The vehicle was also used to develop calibration refinements for improved exhaust emissions and fuel efficiency. Samples of a new GDCI development level engine, called Gen2 GDCI (and developed outside of the first DOE funded project), were also used during Phase 1 to develop refined controller hardware and control algorithms, as well as improved sensors and actuators. The Gen2 engine was then retrofitted into the development vehicle from the ATP1 project and used for controls refinement and calibration of GDCI to achieve improved cold start, drivability and robustness to real world environments and fuel variations. During Phase 2 of the project, Gen3 versions of the GDCI engine were designed and built specifically for this project based on experience from the earlier engines. These engines were designed to be production-like in construction and, when combined with refined control systems and project specific low temperature exhaust aftertreatment, were intended to support the goal of meeting Tier 3 emissions levels at a vehicle level. Though great progress had been made advancing the GDCI combustion process, and despite extensive presentations of our work on the technology at various conferences and in direct meetings with automotive companies, none of the US domestic automotive OEMs had shown an interest in adopting this combustion process as part of their technology planning as the project approached the Go/No-Go point at the end of Budget Period 3. As a result of this low-level of interest both the US Department of Energy and Delphi Technologies received in this technology from domestic auto manufactures, Delphi Technologies elected a ‘No-Go’ status for the gate between Budget Period 3 and Budget Period 4 which terminated the project without spending any Budget Period 4 funds. The project team concluded that the dynamometer mapping of the most recent level of hardware, combined with the vehicle level simulation by Argonne National Laboratory, was sufficient to document the capabilities and potential of the combustion process without spending the time and money to do the controls development and full calibration necessary for a demonstration vehicle.

33 ADVANCED PROPULSION SYSTEMS↗
DOE OSTI2019

An (adjustable RN)-based multi-OLT PON and the dynamic programming algorithm for downstream channel

In this paper, a multiple optical line terminal (OLT) passive optical network (PON) and the dynamic programming algorithm for downstream channel is proposed. By wavelength-selectable module, the adjustable remote node (RN) is constructed. By this adjustable RN, a simple and efficient multi-OLT PON is designed. By the way of first-plan and then-load, optical network unit (ONU) groups are established in order to realize the fast loading of ONUs. Specially, by introducing dynamic programming algorithm, the wavelength bandwidth utilization is maximized. Finally, by the simulation and analysis, the effectiveness of the proposed scheme is demonstrated.

42 ENGINEERING↗
DOE OSTI2024

Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Here, considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Check valve with pilot tube pressure sensing

A check valve's housing has a valve disposed therein to open or close a flow path from the housing's inlet to its outlet based on a difference between pressures at the inlet and outlet. The valve includes a bellows, a poppet, and a spring. The poppet includes a hollow structure extending through the poppet's plate and terminating in a first open end disposed within the bellows, an annular flange between the second face of the poppet's plate and the housing's outlet, and an open-ended tube extending from the annular flange into the housing's outlet. The spring, disposed within the hollow tubular structure, positions the annular flange to seal the outlet when the pressure difference is less than a predetermined amount and compresses when the difference exceeds the predetermined amount wherein the annular flange is unsealed from the housing's outlet to open the flow path.

42 ENGINEERING↗
DOE OSTI2020

Structure and Function of BorB, the Type II Thioesterase from the Borrelidin Biosynthetic Gene Cluster

α/β hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase α/β hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tü4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. Overall, the structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

On the Convergence of Overlapping Schwarz Decomposition for Nonlinear Optimal Control

Here, we study the convergence properties of an overlapping Schwarz decomposition algorithm for solving nonlinear optimal control problems (OCPs). The algorithm decomposes the time domain into a set of overlapping subdomains, and solves all subproblems defined over subdomains in parallel. The convergence is attained by updating primal-dual information at the boundaries of overlapping subdomains. We show that the algorithm exhibits local linear convergence, and that the convergence rate improves exponentially with the overlap size. We also establish global convergence results for a general quadratic programming, which enables the application of the Schwarz scheme inside second-order optimization algorithms (e.g., sequential quadratic programming). The theoretical foundation of our convergence analysis is a sensitivity result of nonlinear OCPs, which we call "exponential decay of sensitivity" (EDS). Intuitively, EDS states that the impact of perturbations at domain boundaries (i.e., initial and terminal time) on the solution decays exponentially as one moves into the domain. Here, we expand a previous analysis available in the literature by showing that EDS holds for both primal and dual solutions of nonlinear OCPs, under uniform second-order sufficient condition, controllability condition, and boundedness condition. We conduct experiments with a quadrotor motion planning problem and a partial differential equations (PDE) control problem to validate our theory, and show that the approach is significantly more efficient than alternating direction method of multipliers and as efficient as the centralized interior-point solver.

42 ENGINEERING↗
DOE OSTI2020

High Altitude Electromagnetic Pulse Testing of Photovoltaic Modules

This report details the test setup, process, and results for radiated susceptibility testing of multicrystalline silicon photovoltaic (PV) modules as part of the EMP-Resilient Electric Grid Grand Challenge Laboratory Directed Research and Development (LDRD) project at Sandia National Laboratories. Testing was conducted over October 10-17, 2019, where 8 photovoltaic modules were exposed to E1 transient pulses with peak field levels up to 100 kV/m. Modules were terminated in a resistive load representing connected components. State of health testing conducted via I-V curve tracing of the photovoltaic modules showed no observable loss of device function due to large electric field transients. Differential mode currents were measured on the order of 10's of amps for up to a microsecond following the radiated field pulse. Common mode currents took the form of a damped sinusoid with a maximum peak of 10's to 100's of amps with a resonance near 60 MHz.

14 SOLAR ENERGY↗
DOE OSTI2021

Grafting nanometer metal/oxide interface towards enhanced low-temperature acetylene semi-hydrogenation

Metal/oxide interface is of fundamental significance to heterogeneous catalysis because the seemingly “inert” oxide support can modulate the morphology, atomic and electronic structures of the metal catalyst through the interface. The interfacial effects are well studied over a bulk oxide support but remain elusive for nanometer-sized systems like clusters, arising from the challenges associated with chemical synthesis and structural elucidation of such hybrid clusters. We hereby demonstrate the essential catalytic roles of a nanometer metal/oxide interface constructed by a hybrid Pd/Bi 2 O 3 cluster ensemble, which is fabricated by a facile stepwise photochemical method. The Pd/Bi 2 O 3 cluster, of which the hybrid structure is elucidated by combined electron microscopy and microanalysis, features a small Pd-Pd coordination number and more importantly a Pd-Bi spatial correlation ascribed to the heterografting between Pd and Bi terminated Bi 2 O 3 clusters. The intra-cluster electron transfer towards Pd across the as-formed nanometer metal/oxide interface significantly weakens the ethylene adsorption without compromising the hydrogen activation. As a result, a 91% selectivity of ethylene and 90% conversion of acetylene can be achieved in a front-end hydrogenation process with a temperature as low as 44 °C.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Fast Proton Insertion in Layered H 2 W 2 O 7 via Selective Etching of an Aurivillius Phase

H 2 W 2 O 7 , a metastable material synthesized via selective etching of the Aurivillius-related Bi 2 W 2 O 9 , is shown as an electrode for high power proton-based energy storage. Comprehensive structural characterization is performed to obtain a high-fidelity crystal structure of H 2 W 2 O 7 using an iterative approach that combines X-ray diffraction, neutron pair distribution function, scanning transmission electron microscopy, Raman spectroscopy, and density functional theory modeling. Electrochemical characterization shows a capacity retention of ≈80% at 1000 mV s –1 (1.5-s charge/discharge time) as compared to 1 mV s –1 (≈16-min charge/discharge time) with cyclability for over 100 000 cycles. Energetics from density functional theory calculations indicate that proton storage occurs at the terminal oxygen sites within the hydrated interlayer. Last, optical micrographs collected during in situ Raman spectroscopy show reversible, multicolor electrochromism, with color changes from pale yellow to blue, purple, and last, orange as a function of proton content. These results highlight the use of selective etching of layered perovskites for the synthesis of metastable transition metal oxide materials and the use of H 2 W 2 O 7 as an anode material for proton-based energy storage or electrochromic applications.

25 ENERGY STORAGE↗
DOE OSTI2021

An experimental and kinetic modeling study of NO x sensitization on methane autoignition and oxidation

An experimental and kinetic modeling study of the influence of NO x (i.e. NO 2 , NO and N 2 O) addition on the ignition behavior of methane/‘air’ mixtures is performed. Ignition delay time measurements are taken in a rapid compression machine (RCM) and in a shock tube (ST) at temperatures and pressures ranging from 900–1500 K and 1.5–3.0 MPa, respectively for equivalence ratios of 0.5–2.0 in ‘air’. The conditions chosen are relevant to spark ignition and homogeneous charge compression ignition engine operating conditions where exhaust gas recirculation can potentially add NO x to the premixed charge. The RCM measurements show that the addition of 200 ppm NO 2 to the stoichiometric CH 4 /oxidizer mixture results in a factor of three increase in reactivity compared to the baseline case without NOx for temperatures in the range 600–1000 K. However, adding up to 1000 ppm N 2 O does not show any appreciable effect on the measurements. The promoting effect of NO 2 was found to increase with temperature in the range 950–1150 K, while the sensitization effect decreases at higher pressures. The experimental results measured are simulated using NUIGMech1.2 comprising an updated NO x sub-chemistry in this work. A kinetic analysis indicates that the competition between the reactions ĊH 3 + NO 2 ↔ CH 3 Ȯ + NO and ĊH 3 + NO 2 (+M) ↔ CH 3 NO 2 (+M), the former being a propagation reaction and the latter being a termination reaction governs NO x sensitization on CH 4 ignition. Recent calculations by Matsugi and Shiina (A. Matsugi, H. Shiina, J. Phys. Chem. A. 121 (2017) 4218–4224) for the nitromethane formation reaction CH 3 + NO 2 (+M) ↔ CH 3 NO 2 (+M), together with the recently calculated rate constants for HONO/HNO 2 reactions significantly improve ignition delay time predictions in the temperature range 600–1000 K. Furthermore, the experiments with NO addition reveal a non-monotonous sensitization impact on CH 4 ignition at lower temperatures with NO initially acting as an inhibitor at low NO concentrations and then as a promoter as NO concentrations increase in the mixture. This non-monotonous trend is attributed to the role of the chain-termination reaction ĊH 3 + NO 2 (+M) ↔ CH 3 NO 2 (+M) and the impact of NO on the transition to the chain-branching steps CH 2 O + HȮ 2 ↔ HĊO + H 2 O 2 , H 2 O 2 (+M) ↔ ȮH + ȮH (+M), HĊO ↔ CO + Ḣ followed by CO + O 2 ↔ CO 2 + Ö and Ḣ + O 2 ↔ Ö + ȮH. NUIGMech1.2 is systematically validated against the new ignition delay measurements taken here together with species measurements and high temperature ignition delay time data available in the literature for CH 4 /oxidizer mixtures diluted with NO 2 /N 2 O/NO and is observed to accurately capture the sensitization trends.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli : bioinformatic and experimental approaches

Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO 2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO 2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗