Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex: e1 and e2 substrate specificty, e1 carboligase activity, and e2 interchain succinyl transfer

Escherichia coli (E. coli) 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc) contains three components: a thiamin di phosphate (ThD P) dependent 2-oxogl utarate dehydrogenase (E1 o), a di hydrol i poylsucci nyl transferase (E2o), and a di hydrol i poyl dehydrogenase (E3). The first two components carry out the principal reactions for succinyl CoA formation while the third one reoxidizes dihydrolipoamide to lipoamide. This mechanism is similar to other 2-oxoacid dehydrogenase complexes, including pyruvate dehydrogenases (PDHc) and branched-chain dehydrogenases. E1o of the E. coli OGDHc was engineered to accept unnatural substrates. The natural substrate for E1 o is 2-oxogl utarate (2-OG) and the enzyme was engineered to accept substrates lacking the 5-carboxylate group, 2-oxovalerate (2-OV). E1o was subjected to saturation mutagenesis at H is260 and H is298. The H is298Asp E1 o variant was identified in the screen to accept the unnatural substrate. In addition, it was discovered that His260 is required for substrate recognition, but His298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the “gatekeeper” for specificity for these two multienzyme complexes in bacteria, E1 p for pyruvate but E2o for 2-OG. Although His298Asp E1o accepted 2-OV, reconstitution of the variant with E2o and E3 did not generate NADH in the overall reaction using 2-OV. Hence, the reaction may be hindered in the E2o component. E2o consists of an amino-terminal lipoyl domain (E2oLD, 12 kDa), followed by a peripheral subunit binding domain (4 kDa) and a succinyltransferase domain (E2oCD, 28 kDa). There are two possibilities for the failure to form NADH. Reductive acylation is not occurring in the E2oLD or acyl transfer to CoA is not taking place in E2oCD. His298Asp E1o, E2oLD, and 2-OV form butyrylated E2oLD, which was shown by electrospray ionization mass spectrometry. Therefore, the E2oCD domain necessitates optimization to produce acyl-CoA derivatives. Succinyl transfer to the CD domain may occur through an intrachain or interchain pathway. The E2oLD and E2o with a lysine to alanine substitution at position 43 (E2oK43A) were created by site directed mutagenesis. It is clearly shown that E2oLD was capable of rescuing the crippled E2oK43A variant by measuring the NADH production in the overall reaction. Therefore, an interchain mechanism is likely between two different E2o subunits. ThDP-dependent enzymes have the potential to be used for chemical synthesis. These enzymes share a common feature in that they catalyze carboligase reactions. E1o catalyzed carboligation products with a variety of substrates and acceptors that vary in the size and functional groups. Structures of the products were confirmed with NMR. In addition, high enantiomeric excess (ee) values were found for the products as shown by chiral gas chromatography and CD spectroscopy. Finally, it was shown that E1o is capable of forming stable esters. This is important because when the carboligase reactions produce 0-ketoacids, these products are unstable and prone to decarboxylation

Comparison of optical density, total cell protein, and number of viable cells (via fluorescent straining) as measures of microbial growth kinetics in suspended and biofilm cultures during biodegradation of naphthalene

Most studies on bacterial growth kinetics have been dependent on theoretical modeling with general biomass measurements using either dry weight or optical density (OD), without distinguishing live from dead bacteria or debris. As a result, there remains considerable uncertainty in reliably predicting rates of biodegradation for design of treatment processes for environmental pollutants. This research focused on measurement of bacterial growth rates and activities in suspended cultures and biofilms using Pseudomonas putida (ATCC 17484) for biodegradation of naphthalene. As expected, the rates of biodegradation differed between suspended and immobilized cultures. A comparison was made of the impact of three biomass measures: optical density, total cell protein, and living cell number on the calculated rate of naphthalene disappearance. Living cell number was determined by a fluorescent staining technique and use of epifluorescence microscopy. More than 90% of total cells remained viable over the course of each experiment (35 to 54 hours). All three techniques experienced difficulties reconciling calculated values of biomass growth and naphthalene disappearance. This was considered to be a consequence of the production of intermediate products detected in the chromatograms, and possibly adsorption and subsequent release of naphthalene, which resulted in a lag time between the isappearance of naphthalene and the appearance of biomass. Inclusion of a lag time in the integrated Monod expression improved the agreement between experimental and calculated values of biomass and naphthalene concentrations. However, further improvements will require more detailed kinetics of the actual biochemical pathway

Bio-oxidation of a model VOC in air

This study was performed to find a satisfactory regime of operation for the vapor phase bio-oxidation of ethyl alcohol, a model volatile organic compound (VOC), in a specially designed bioreactor. Ethanol was selected as a model compound representing bakery oven gas pollution. A spirally wound bioreactor module was used within which was immobilized a mixed bacterial culture from aerobic sludge. The activated sludge from municipal wastewater treatment plants readily attached with no pretreatment on the surface of the spiral biosupport which is a polymeric sheet. The parameters studied were air flow rate and inlet concentrations of ethanol. Ethanol was injected, along with air, into a water reservoir prior to being fed into the bioreactor. The vapor and liquid concentrations in the reservoir were measured continuously and reached an equilibrium state. The reaction rates for all runs were determined. An optimal vapor temperature was observed for the environment of the microorganisms. As expected, the reaction rate varied with air flow rate and vapor temperature. An optimal air flow rate which was used for the action of microorganisms with gaseous ethyl alcohol and oxygen was 20 L/min (retention time:1.45 min). At this flow rate, the desirable vapor temperature in the reservoir was between 27 °C and 30 °C. At 20 L/min of air flow, a local maximum reaction rate was maintained at about 44 to 50 mg of ethanol per minute for this 28 hour run at this feed injection level. The vapor concentration at the inlet in typical runs from this series at this flow rate reached equilibrium levels ranging from about 1,000 ppmv to 1,700 ppmv within the first four hours of the 28 hour runs. The air flow rates for this series ranged from 7.52 to 40 L/min, while the total amount of ethanol fed to the system per minute was kept constant. At higher inlet feed concentrations, the reaction rates increased. For this series, at 2.34 L/min of air flow (retention time: 12.35 min), the maximum inlet vapor concentration reached about 7,000 ppmv within 6 hours. The removal efficiency was 99 percent, equivalent to about 30 mg of ethanol/min due to the low air flow rate. Also, after a 6 hour run at 7.52 L/min of air flow (retention time: 3.84 min), about 6,000 ppmv at the inlet was converted to 24 ppmv at the outlet. The removal efficiency was 99 percent, equivalent to about 87 mg of ethanol/min. This is about 3 times the corresponding rate at 2.34 L/min of air flow

Nonlinear dynamical tunneling of optical whispering gallery modes in the presence of a Kerr nonlinearity

The effect of a Kerr nonlinearity on dynamical tunneling is studied, using coupled whispering gallery modes in an optical microcavity. The model system that we have chosen is the 'add-drop filter', which comprises an optical microcavity and two waveguide coupled to the cavity. Due to the evanescent field's scattering on the waveguide, the whispering gallery modes in the microcavity form doublets, which manifest themselves as splittings in the spectrum. As these doublets can be regarded as a spectral feature of dynamical tunneling between two different dynamical states with a spatial overlap, the effect of a Kerr nonlinearity on the doublets is numerically investigated in the more general context of the relationship between cubic nonlinearity and dynamical tunneling. Within the numerical realization of the model system, it is observed that the doublets shows a bistable transition in its transmission curve as the Kerr-nonlinearity in the cavity is increased. At the same time, one rotational mode gets dominant over the other one in the transmission, since the two states in the doublet have uneven linewidths. By using coupled mode theory, the underlying mode dynamics of the phenomena is theoretically modelled and clarified.Comment: 7 pages, 5 figure

Faraday instability and subthreshold Faraday waves: surface waves emitted by walkers

A walker is a fluid entity comprising a bouncing droplet coupled to the waves that it generates at the surface of a vibrated bath. Thanks to this coupling, walkers exhibit a series of wave-particle features formerly thought to be exclusive to the quantum realm. In this paper, we derive a model of the Faraday surface waves generated by an impact upon a vertically vibrated liquid surface. We then particularise this theoretical framework to the case of forcing slightly below the Faraday instability threshold. Among others, this theory yields a rationale for the dependence of the wave amplitude to the phase of impact, as well as the characteristic timescale and length scale of viscous damping. The theory is validated with experiments of bead impact on a vibrated bath. We finally discuss implications of these results for the analogy between walkers and quantum particles

Fragility, Stokes-Einstein violation, and correlated local excitations in a coarse-grained model of an ionic liquid

Dynamics of a coarse-grained model for the room-temperature ionic liquid, 1-ethyl-3-methylimidazolium hexafluorophosphate, couched in the united-atom site representation are studied via molecular dynamics simulations. The dynamically heterogeneous behavior of the model resembles that of fragile supercooled liquids. At or close to room temperature, the model ionic liquid exhibits slow dynamics, characterized by nonexponential structural relaxation and subdiffusive behavior. The structural relaxation time, closely related to the viscosity, shows a super-Arrhenius behavior. Local excitations, defined as displacement of an ion exceeding a threshold distance, are found to be mainly responsible for structural relaxation in the alternating structure of cations and anions. As the temperature is lowered, excitations become progressively more correlated. This results in the decoupling of exchange and persistence times, reflecting a violation of the Stokes-Einstein relation.Comment: Published on the Phys. Chem. Chem. Phys. websit

Bohmian trajectories for the half-line barrier

Bohmian trajectories are considered for a particle that is free (i.e. the potential energy is zero), except for a half-line barrier. On the barrier, both Dirichlet and Neumann boundary conditions are considered. The half-line barrier yields one of the simplest cases of diffraction. Using the exact time-dependent propagator found by Schulman, the trajectories are computed numerically for different initial Gaussian wave packets. In particular, it is found that different boundary conditions may lead to qualitatively different sets of trajectories. In the Dirichlet case, the particles tend to be more strongly repelled. The case of an incoming plane wave is also considered. The corresponding Bohmian trajectories are compared with the trajectories of an oil drop hopping on the surface of a vibrating bath

A Study on the improvement of military manpower system of ROK armed forces through introduction of partial volunteer force system

Thesis(Master) --KDI School:Master of Public Policy,2017This paper is designed to explore the military service system to maintain effective military service under low-fertility conditions in changing circumstances. South Korea has long since become an ultra-low birth rate country all over the world. Therefore, it is expected that it will be difficult to maintain the current military scale due to the decrease of young people who can join the military in the near future. Despite these circumstances, the military service period was shortened to 21 months due to many factors such as the issue of wartime operational control, political issues (presidential election pledges) and defense reform plans. In addition, despite the low birth rate problem, the social demand for shortening the military service period and introducing the volunteer force system is expected to be larger in the future. The Korean peninsula is the world's only divided nation, and the security threat of the Korean Peninsula has never been reduced due to the development of nuclear weapons on the North. Also, North Korea, which is confronted with South Korea, is a military power that maintains more than one million soldiers. Considering the North Korean threat, political and economic problems, military factors, and population problems, I sought to find the most appropriate conscription system that Korea could apply at this time. This study will estimate the quantitative of shortage troops in the future. And through introduction of partially volunteer force, this study will suggest the better way to change troops structure more efficiently.CHAPTER Ⅰ. Introduction CHAPTER Ⅱ. Review of related literature CHAPTER Ⅲ. Population structure of Korea CHAPTER Ⅳ. Future demand for national defence CHAPTER Ⅴ. Optimal Plan to Replace the Current Conscription System CHAPTER Ⅵ. Factors to consider when restructuring the workforce CHAPTER Ⅶ. ConclusionmasterpublishedJeong-rock SHIM