Production of biodiesel using a continuous gas-liquid reactor

A novel continuous reactor process has been developed for the production of biodiesel from fats and oils. The key feature of the process is its ability to operate continuously with a high reaction rate, potentially requiring less post reaction cleaning and product/reactant separation than currently established processes. This was achieved by atomising the heated oil/fat and then spraying it into a reaction chamber filled with methanol vapor in a counter current flow arrangement. This allows the continuous separation of product and the excess methanol stream in the reactor. The overall conversion based on a single cycle of this process has been between 50% and 96% of the feed stock materials. Conversions of 94-96% were achieved while operating with 5-7 g of sodium methoxide/L of methanol at methanol flow rate of 17.2 L/h and oil flow rate of 10 L/h. Additional variations in the reactant stoichiometry (i.e. reactant flow rates), catalyst type/concentration, and reaction temperature on the overall product conversion were investigated.     

Consistent Estimation of Gibbs Energy Using Component Contributions

Standard Gibbs energies of reactions are increasingly being used in metabolic modeling for applying thermodynamic constraints on reaction rates, metabolite concentrations and kinetic parameters. The increasing scope and diversity of metabolic models has led scientists to look for genome-scale solutions that can estimate the standard Gibbs energy of all the reactions in metabolism. Group contribution methods greatly increase coverage, albeit at the price of decreased precision. We present here a way to combine the estimations of group contribution with the more accurate reactant contributions by decomposing each reaction into two parts and applying one of the methods on each of them. This method gives priority to the reactant contributions over group contributions while guaranteeing that all estimations will be consistent, i.e. will not violate the first law of thermodynamics. We show that there is a significant increase in the accuracy of our estimations compared to standard group contribution. Specifically, our cross-validation results show an 80% reduction in the median absolute residual for reactions that can be derived by reactant contributions only. We provide the full framework and source code for deriving estimates of standard reaction Gibbs energy, as well as confidence intervals, and believe this will facilitate the wide use of thermodynamic data for a better understanding of metabolism.     

Transport effects on surface–volume biological reactions

Many cellular reactions involve a reactant in solution binding to or dissociating from a reactant confined to a surface. This is true as well for a BIAcore, an optical biosensor that is widely used to study the interaction of biomolecules. In the flow cell of this instrument, one of the reactants is immobilized on a flat sensor surface while the other reactant flows past the surface. Both diffusion and convection play important roles in bringing the reactants into contact. Usually BIAcore binding data are analyzed using well known expressions that are valid only in the reaction-limited case when the Damk?hler number Da is small. Asymptotic and singular perturbation techniques are used to analyze dissociation of the bound state when Da is small and O(1). Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. In addition, effective rate constants are derived that illustrate the effects of transport on the measured rate constants. All these expressions provide a direct way to estimate the rate constants from BIAcore binding data.     

Construction of a microprocessor-controlled pulsed quench-flow apparatus for the study of fast chemical and biochemical reactions

The construction and operation of a microprocessor-controlled quenched-flow machine are described. Two sets of syringes are moved by high-torque stepping motors to achieve any desired mixing scheme. The dead time of the instrument is pulsed quench-flow operation is of the order of 10 ms. The fastest possible reaction time is 12 ms with the reaction chamber used here; this might be extended downward with other reaction chambers. The reactant volume required is low; 1 ml of solution for each reactant is sufficient for more than 40 kinetic measurements. The machine is tested by alkaline hydrolysis of 2,4-dinitrophenylacetate and used in the investigation of the reaction mechanism of the EcoRI restriction endonuclease and of GTPase activity of ribosomes.     

Transport and kinetic processes underlying biomolecular interactions in the BIACORE optical biosensor

The transport and kinetic processes describing biomolecular interactions in the BIACORE optical biosensor have been studied with the help of a mathematical model. In comparison to previous models, the model presented here couples, for the first time, transport phenomena in the flow channel with hindered diffusive transport and reactions inside the hydrogel. Simulated experiments based on this model, and two simpler models extant in the literature, are used to identify cases under which the detailed model is essential for accurate prediction of kinetic parameters. It is shown that this model can substantially improve the accuracy of kinetic parameter estimation when transport limitations in the flow channel and/or the hydrogel significantly influence the observed instrument response curves. The model can extend the range of the instrument's applicability to higher concentrations of immobilized species within the hydrogel. It can also be used for accurate design of experiments with the purpose of minimizing errors in the estimation of the kinetic parameters.     

BIACORE J: a new platform for routine biomolecular interaction analysis

SPR biosensor technology continues to evolve. The recently released platform from Biacore AB (Uppsala, Sweden), BIACORE J, is designed for the routine analysis of biomolecular interactions. Using an antibody-protein A and a ligand-receptor system, we demonstrate the utility of BIACORE J in determining active concentration and binding affinities. The results from these studies illustrate the high sensitivity of the instrument and its ability to generate reproducible binding responses. The BIACORE J is easy to operate and useful in diverse applications, making SPR technology widely accessible as a research tool.     

Metabolic Pathfinding Using RPAIR Annotation

Metabolic databases contain information about thousands of small molecules and reactions, which can be represented as networks. In the context of metabolic reconstruction, pathways can be inferred by searching optimal paths in such networks. A recurrent problem is the presence of pool metabolites (e.g., water, energy carriers, and cofactors), which are connected to hundreds of reactions, thus establishing irrelevant shortcuts between nodes of the network. One solution to this problem relies on weighted networks to penalize highly connected compounds. A more refined solution takes the chemical structure of reactants into account in order to differentiate between side and main compounds of a reaction. Thanks to an intensive annotation effort at KEGG, decompositions of reactions into reactant pairs (RPAIR) categorized by their role (main, trans, cofac, ligase, and leave) are now available.The goal of this article is to evaluate the impact of RPAIR data on pathfinding in metabolic networks. To this end, we measure the impact of different parameters concerning the construction of the metabolic network: mapping of reactions and reactant pairs onto a graph, use of selected categories of reactant pairs, weighting schemes for compounds and reactions, removal of highly connected metabolites, and reaction directionality. In total, we tested 104 combinations of parameters and identified their optimal values for pathfinding on the basis of 55 reference pathways from three organisms.The best-performing metabolic network combines the biochemical knowledge encoded by KEGG RPAIR with a weighting scheme penalizing highly connected compounds. With this network, we could recover reference pathways from Escherichia coli with an average accuracy of 93% (32 pathways), from Saccharomyces cerevisiae with an average accuracy of 66% (11 pathways), and from humans with an average accuracy of 70% (12 pathways). Our pathfinding approach is available as part of the Network Analysis Tools.     

A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.     

Calculation of effective diffusivities for biofilms and tissues.

In this study we describe a scheme for numerically calculating the effective diffusivity of cellular systems such as biofilms and tissues. This work extends previous studies in which we developed the macroscale representations of the transport equations for cellular systems based on the subcellular-scale transport and reaction processes. A finite-difference model is used to predict the effective diffusivity of a cellular system on the basis of the subcellular-scale geometry and transport parameters. The effective diffusivity is predicted for a complex three-dimensional structure that is based on laboratory observations of a biofilm, and these numerical predictions are compared with predictions from a simple analytical solution and with experimental data. Our results indicate that, under many practical circumstances, the simple analytical solution can be used to provide reasonable estimates of the effective diffusivity.     

The effect of a receptor layer on the measurement of rate constants

Many cellular reactions involve a reactant in solution binding to or dissociating from a reactant attached to a surface. Most studies assume that the reactions occur on this surface, when in actuality the receptors usually lie in a thin layer on top of it. The effect of this layer is considered, particularly as it relates to the BIAcore™ measurement device, though the results are applicable to biological systems. A dimensionless parameter measuring the strength of the effect of the receptor layer is found. Asymptotic and singular perturbation techniques are used to analyse association and dissociation kinetics, though the effect of the receptor layer need not be small. Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. In addition, effective rate constants are derived that illustrate the combined effects of transport and the receptor layer on the measured rate constants. All these expressions provide a direct way to estimate rate constants from BIAcore™ binding data.     

Kinetic studies of RNA-protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.     

Fermentation data analysis and state estimation in the presence of incomplete mass balance

A method is developed for identifying measurement errors and estimating fermentation states in the presence of unidentified reactant or product. Unlike conventional approaches using elemental balances, this method employs an empirically determined basis, which can tolerate unidentified reaction species. The essence of this approach is derived from the concept of reaction subspace and the technique of singular value decomposition. It is shown that the subspace determined via singular value decomposition of multiple experimental data provides an empirical basis for identifying measurement errors. The same approach is applied to fermentation state estimation. Via the formulation of the reaction subspace, the sensitivity of state estimates to measurement errors is quantified in terms of a dimensionless quantity, maximum error gain (MEG). It is shown that using the empirically determined subspace, one can circumvent the problem of unidentified reaction species, meanwhile reducing the sensitivity of the estimates.     

Mass transfer effects on DNA hybridization in a flow-through microarray

The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization.     

A Thermodynamic Characterization of the Binding of Thrombin Inhibitors to Human Thrombin, Combining Biosensor Technology, Stopped-Flow Spectrophotometry, and Microcalorimetry

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

Analysis of coupled bimolecular reaction kinetics and diffusion by two-color fluorescence correlation spectroscopy: enhanced resolution of kinetics by resonance energy transfer

In two-color fluorescence correlation spectroscopy (TCFCS), the fluorescence intensities of two fluorescently-labeled species are cross-correlated over time and can be used to identify static and dynamic interactions. Generally, fluorophore labels are chosen that do not undergo F?rster resonance energy transfer (FRET). Here, a general TCFCS theory is presented that accounts for the possibility of FRET between reactants in the reversible bimolecular reaction, [reaction: see text] where k(f) and k(b) are forward and reverse rate constants, respectively (dissociation constant K(d) = k(b)/k(f)). Using this theory, we systematically investigated the influence on the correlation function of FRET, reaction rates, reactant concentrations, diffusion, and component visibility. For reactants of comparable size and an energy-transfer efficiency of approximately 90%, experimentally measurable cross-correlation functions should be sensitive to reaction kinetics for K(d) > 10(-8) M and k(f) >or= approximately 10(7) M(-1)s(-1). Measured auto-correlation functions corresponding to donor and acceptor labels are generally less sensitive to reaction kinetics, although for the acceptor, this sensitivity increases as the visibility of the donor increases relative to the acceptor. In the absence of FRET or a significant hydrodynamic difference between reactant species, there is little effect of reaction kinetics on the shape of auto- and cross-correlation functions. Our results suggest that a subset of biologically relevant association-dissociation kinetics can be measured by TCFCS and that FRET can be advantageous in enhancing these effects.     

The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore

Accurate estimation of biomolecular reaction rates from binding data, when ligands in solution bind to receptors on the surfaces of cells or biosensors, requires an understanding of the contributions of both molecular transport and reaction. Efficient estimation of parameters requires relatively simple models. In this review, we give conditions under which various transport effects are negligible and identify simple binding models that incorporate the effects of transport, when transport cannot be neglected. We consider effects of diffusion of ligands to cell or biosensor surfaces, flow in a BIAcore biosensor, and distribution of receptors in a dextran layer above the sensor surface. We also give conditions under which soluble receptors can be expected to compete effectively with surface-bound receptors.     

Anterograde Transport of Herpes Simplex Virus Proteins in Axons of Peripheral Human Fetal Neurons: an Immunoelectron Microscopy Study

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.     

Chemical reactions and membranes: A macroscopic basis for active transport II. Non-linear aspects

The physical principles that material and charge are conserved provide a basis for the design of a membrane capable of performing active ion transport in which the connection between “metabolic energy” input and the ion transport process itself is electrical rather than material. Molecular interactions between components in this system are irrelevant to its function. A second model built on the same principles performs active ion transport in a statically symmetrical membrane. The basis of its operation is a weakly stable unsymmetrical concentration profile arising from an enzyme-catalyzed reaction occurring within the membrane. The function of this membrane is irreversibly terminated (“killed”) by interference either with intramembrane concentration gradients or with the reactions which maintain them. Hence, any attempt to study this system by breaking it apart destroys the basis of its function. The existence of these models reveals the logical insufficiency of the molecular biologist's approach to understanding the basis of active transport: Neither the experimental techniques for structure determination (disruption, purification, characterization, and reconstitution) nor the fundamental question of that discipline (What is the molecular connection between &*|… ?) of the molecular biologist are applicable to the study or interpretation of these model systems. While the model systems are artificial, they incorporate only widely applicable concepts of physical chemistry and biochemical kinetics. The is no reason for excluding such mechanisms in natural membrane transport systems. If they are present, then more effective strategies of investigation will be required.     

Development and application of surface plasmon resonance-based biosensors for the detection of cell-ligand interactions

Surface plasmon resonance (SPR)-based biosensors were investigated with a view to providing a portable, inexpensive alternative to existing technologies for "real-time" biomolecular interaction analysis of whole cell-ligand interactions. A fiber optic SPR-based (FOSPR) biosensor, employing wavelength-dependent SPR, was constructed to enable continuous real-time data acquisition. In addition, a commercially available integrated angle-dependent SPR-based refractometer (ISPR) was modified to facilitate biosensing applications. Solid-phase detection of whole red blood cells (RBCs) using affinity-captured blood group specific antibodies was demonstrated using the BIACORE 1000, BIACORE Probe, FOSPR, and ISPR sensors. Nonspecific binding of RBCs to the hydrogel-based biointerface was negligible. However, the background noise level of the FOSPR-based biosensor was approximately 25-fold higher than that of the widely used BIACORE 1000 system while that of the ISPR-based biosensor was over 100-fold higher. Nevertheless, the FOSPR biosensor was suitable for the analysis of macromolecular analytes contained in crude matrices.