Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes

‘Ene’-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The b,b-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the a,b-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56% and poor enantioselectivity (16% ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of ‘ene’-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds

The putative mevalonate diphosphate decarboxylase from Picrophilus torridus is in reality a mevalonate-3-kinase with high potential for bioproduction of isobutene

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway.MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme’s potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min-1 g cells-1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min-1 mg protein-1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway

Formation of topological defects in gauge field theories

When a symmetry gets spontaneously broken in a phase transition, topological defects are typically formed. The theoretical picture of how this happens in a breakdown of a global symmetry, the Kibble-Zurek mechanism, is well established and has been tested in various condensed matter experiments. However, from the viewpoint of particle physics and cosmology, gauge field theories are more relevant than global theories. In recent years, there have been significant advances in the theory of defect formation in gauge field theories, which make precise predictions possible, and in experimental techniques that can be used to test these predictions in superconductor experiments. This opens up the possibility of carrying out relatively simple and controlled experiments, in which the non-equilibrium phase transition dynamics of gauge field theories can be studied. This will have a significant impact on our understanding of phase transitions in the early universe and in heavy ion collider experiments. In this paper, I review the current status of the theory and the experiments in which it can be tested.Comment: Review article, 43 pages, 7 figures. Minor changes, some references added. Final version to appear in IJMP

Counting defects with the two-point correlator

We study how topological defects manifest themselves in the equal-time two-point field correlator. We consider a scalar field with Z_2 symmetry in 1, 2 and 3 spatial dimensions, allowing for kinks, domain lines and domain walls, respectively. Using numerical lattice simulations, we find that in any number of dimensions, the correlator in momentum space is to a very good approximation the product of two factors, one describing the spatial distribution of the defects and the other describing the defect shape. When the defects are produced by the Kibble mechanism, the former has a universal form as a function of k/n, which we determine numerically. This signature makes it possible to determine the kink density from the field correlator without having to resort to the Gaussian approximation. This is essential when studying field dynamics with methods relying only on correlators (Schwinger-Dyson, 2PI).Comment: 11 pages, 7 figures

Defect Formation and Critical Dynamics in the Early Universe

We study the nonequilibrium dynamics leading to the formation of topological defects in a symmetry-breaking phase transition of a quantum scalar field with \lambda\Phi^4 self-interaction in a spatially flat, radiation-dominated Friedmann-Robertson-Walker Universe. The quantum field is initially in a finite-temperature symmetry-restored state and the phase transition develops as the Universe expands and cools. We present a first-principles, microscopic approach in which the nonperturbative, nonequilibrium dynamics of the quantum field is derived from the two-loop, two-particle-irreducible closed-time-path effective action. We numerically solve the dynamical equations for the two-point function and we identify signatures of topological defects in the infrared portion of the momentum-space power spectrum. We find that the density of topological defects formed after the phase transition scales as a power law with the expansion rate of the Universe. We calculate the equilibrium critical exponents of the correlation length and relaxation time for this model and show that the power law exponent of the defect density, for both overdamped and underdamped evolution, is in good agreement with the "freeze-out" scenario of Zurek. We introduce an analytic dynamical model, valid near the critical point, that exhibits the same power law scaling of the defect density with the quench rate. By incorporating the realistic quench of the expanding Universe, our approach illuminates the dynamical mechanisms important for topological defect formation. The observed power law scaling of the defect density with the quench rate, observered here in a quantum field theory context, provides evidence for the "freeze-out" scenario in three spatial dimensions.Comment: 31 pages, RevTex, 8 figures in EPS forma

A Grand Canonical Ensemble Approach to the Thermodynamic Properties of the Nucleon in the Quark-Gluon Coupling Model

In this paper, we put forward a way to study the nucleon's thermodynamic properties such as its temperature, entropy and so on, without inputting any free parameters by human hand, even the nucleon's mass and radius. First we use the Lagrangian density of the quark gluon coupling fields to deduce the Dirac Equation of the quarks confined in the gluon fields. By boundary conditions we solve the wave functions and energy eigenvalues of the quarks, and thus get energy-momentum tensor, nucleon mass, and density of states. Then we utilize a hybrid grand canonical ensemble, to generate the temperature and chemical potentials of quarks, antiquarks of three flovars by the four conservation laws of the energy and the valence quark numbers, after which, all other thermodynamic properties are known. The only seemed free paremeter, the nucleon radius is finally determined by the grand potential minimal principle.Comment: 5 pages, LaTe

Electron affinities of the first- and second- row atoms: benchmark ab initio and density functional calculations

A benchmark ab initio and density functional (DFT) study has been carried out on the electron affinities of the first- and second-row atoms. The ab initio study involves basis sets of $spdfgh$ and $spdfghi$ quality, extrapolations to the 1-particle basis set limit, and a combination of the CCSD(T), CCSDT, and full CI electron correlation methods. Scalar relativistic and spin-orbit coupling effects were taken into account. On average, the best ab initio results agree to better than 0.001 eV with the most recent experimental results. Correcting for imperfections in the CCSD(T) method improves the mean absolute error by an order of magnitude, while for accurate results on the second-row atoms inclusion of relativistic corrections is essential. The latter are significantly overestimated at the SCF level; for accurate spin-orbit splitting constants of second-row atoms inclusion of (2s,2p) correlation is essential. In the DFT calculations it is found that results for the 1st-row atoms are very sensitive to the exchange functional, while those for second-row atoms are rather more sensitive to the correlation functional. While the LYP correlation functional works best for first-row atoms, its PW91 counterpart appears to be preferable for second-row atoms. Among ``pure DFT'' (nonhybrid) functionals, G96PW91 (Gill 1996 exchange combined with Perdew-Wang 1991 correlation) puts in the best overall performance. The best results overall are obtained with the 1-parameter hybrid modified Perdew-Wang (mPW1) exchange functionals of Adamo and Barone [J. Chem. Phys. {\bf 108}, 664 (1998)], with mPW1LYP yielding the best results for first-row, and mPW1PW91 for second-row atoms. Indications exist that a hybrid of the type $a$ mPW1LYP + $(1-a)$ mPW1PW91 yields better results than either of the constituent functionals.Comment: Phys. Rev. A, in press (revised version, review of issues concerning DFT and electron affinities added

Nonequilibrium Evolution of Correlation Functions: A Canonical Approach

We study nonequilibrium evolution in a self-interacting quantum field theory invariant under space translation only by using a canonical approach based on the recently developed Liouville-von Neumann formalism. The method is first used to obtain the correlation functions both in and beyond the Hartree approximation, for the quantum mechanical analog of the $\phi^{4}$ model. The technique involves representing the Hamiltonian in a Fock basis of annihilation and creation operators. By separating it into a solvable Gaussian part involving quadratic terms and a perturbation of quartic terms, it is possible to find the improved vacuum state to any desired order. The correlation functions for the field theory are then investigated in the Hartree approximation and those beyond the Hartree approximation are obtained by finding the improved vacuum state corrected up to ${\cal O}(\lambda^2)$. These correlation functions take into account next-to-leading and next-to-next-to-leading order effects in the coupling constant. We also use the Heisenberg formalism to obtain the time evolution equations for the equal-time, connected correlation functions beyond the leading order. These equations are derived by including the connected 4-point functions in the hierarchy. The resulting coupled set of equations form a part of infinite hierarchy of coupled equations relating the various connected n-point functions. The connection with other approaches based on the path integral formalism is established and the physical implications of the set of equations are discussed with particular emphasis on thermalization.Comment: Revtex, 32 pages; substantial new material dealing with non-equilibrium evolution beyond Hartree approx. based on the LvN formalism, has been adde