A novel FRET approach for in situ investigation of cellulase–cellulose interaction

A novel real-time in situ detection method for the investigation of cellulase–cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)–cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase–cellulose study using cellulosic fiber.     

An Electron Beam Initiated Fusion Neutron Generator

An experimental scheme is proposed which seems to satisfy all the requirements for use of a high energy electron beam to initiate a thermonuclear plasma. One-dimensional expansion is utilized to obtain confinement times longer than the pulse length of the electron beams. A magnetic field is used to limit the radial heat conductivity, and this magnetic field also serves as a guiding field for the electron beams when they are in the vicinity of the target. Two opposing electron beams are employed and the forces produced by these counterstreaming currents in the overlap region of the beams are sufficient to stop the beams within the target. Estimates made of all the critical factors indicate that beams achievable with current technology can be focused and stopped in T-D targets 6 cm long with densities as low as 1021 cm-3. With a containing magnetic field of 750 kilogauss the containment time of the plasma is sufficiently long so that beam pulse lengths up to 8 × 10-9 sec can be used. Furthermore a positive fusion energy yield relative to the energy delivered to the target is predicted.     

Influence of caffeine and temperature on corrosion-resistance of CoCrMo alloy

The inhibitory activity of caffeine (1,3,7-trimethyl xanthine) on artificial saliva was studied on a CoCrMo alloy using different electrochemical methods: open circuit potential (OCP), potentiodynamic measurements and electrochemical impedance spectroscopy (EIS). The results show that caffeine produces an inhibitory effect on the anodic currents due to its adsorption on the surface of the alloy. Temperature is another parameter with an influence on corrosion processes, so thermodynamic data were obtained from Arrhenius plots and Langmuir adsorption isotherms. The protective action of caffeine is enhanced at high temperatures at OCP, while for potentiodynamic experiments high temperatures block the inhibitory activity of caffeine and the corrosion rate increases. The process may also be studied by a simulation, determining the functional dependence between OCP, corrosion current density (i _corr), corrosion potential (E _corr), breakdown potential (E _bd) and temperature and amount of caffeine in artificial saliva, for Heraenium® CE. The neural network-based methodology applied in this work provides accurate results, thus proving to be an efficient modelling technique.     

Two Decades of Laccases: Advancing Sustainability in the Chemical Industry

Given the current state of environmental affairs and that our future on this planet as we know it is in jeopardy, research and development into greener and more sustainable technologies within the chemical and forest products industries is at its peak. Given the global scale of these industries, the need for environmentally benign practices is propelling new green processes. These challenges are also impacting academic research and our reagents of interest are laccases. These enzymes are employed in a variety of biotechnological applications due to their native function as catalytic oxidants. They are about as green as it gets when it comes to chemical processes, requiring O₂ as their only co‐substrate and producing H₂O as the sole by‐product. The following account will review our twenty year journey on the use of these enzymes within our research group, from their initial use in biobleaching of kraft pulps and for fiber modification within the pulp and paper industry, to their current application as green catalytic oxidants in the field of synthetic organic chemistry.     

Modelling carbon nanotube based biosensor

This paper presents a two-dimensional-in-space mathematical model of an amperometric biosensor based on an enzyme-loaded carbon nanotubes layer deposited on a perforated membrane. The developed model is based on non-linear non-stationary reaction-diffusion equations. By changing input parameters the output results are numerically analysed with a special emphasis to the influence of the geometry and the catalytic activity of the biosensor to its response. The numerical simulation at transition and steady state conditions was carried out using the finite difference technique. The mathematical model and the numerical solution were validated by experimental data. The obtained agreement between the simulation results and experimental data was admissible at different concentrations of the substrate and the mediator.     

Modelling synergistic action of laccase-based biosensor utilizing simultaneous substrates conversion

The response of a laccase-based amperometric biosensor that acts in a synergistic manner was modelled digitally. A mathematical model of the biosensor is based on a system of non-linear reaction diffusion equations. The modelling biosensor comprises three compartments, an enzyme layer, a dialysis membrane and an outer diffusion layer. By changing input parameters the biosensor action was analysed with a special emphasis to the influence of the species concentrations on the synergy of the simultaneous substrates conversion. The digital simulation was carried out using the finite difference technique.     

Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.     

(13)C cell wall enrichment and ionic liquid NMR analysis: progress towards a high-throughput detailed chemical analysis of the whole plant cell wall

The ability to accurately and rapidly measure plant cell wall composition, relative monolignol content and lignin-hemicellulose inter-unit linkage distributions has become essential to efforts centered on reducing the recalcitrance of biomass by genetic engineering. Growing (13)C enriched transgenic plants is a viable route to achieve the high-throughput, detailed chemical analysis of whole plant cell wall before and after pretreatment and microbial or enzymatic utilization by (13)C nuclear magnetic resonance (NMR) in a perdeuterated ionic liquid solvent system not requiring component isolation. 1D (13)C whole cell wall ionic liquid NMR of natural abundant and (13)C enriched corn stover stem samples suggest that a high level of uniform labeling (>97%) can significantly reduce the total NMR experiment times up to ～220 times. Similarly, significant reduction in total NMR experiment time (～39 times) of the (13)C enriched corn stover stem samples for 2D (13)C-(1)H heteronuclear single quantum coherence NMR was found.     

Capillary electrophoretic determination of thiosulfate and its oxidation products

Capillary electrophoresis (CE) was developed for the rapid and simple determination of thiosulfate and its oxidation products such as common polythionates, sulfite and sulfate. Direct and indirect UV detection techniques were investigated. The optimized separations of UV absorbing S2O3(2-), S4O6(2-), S5O6(2-) and S6O6(2-) anions were carried out in 5 mmol l(-1) (NH4)2SO4, 5 mmol l(-1) KH2PO4 electrolyte at pH 5.0, with direct UV detection at 214 nm. All analytes were well resolved in less than 4 min. Analysis of S2O3(2-), S4O6(2-), SO4(2-) and SO3(2-) ions can be performed in 5 mmol l(-1) H2CrO4, 1 mmol l(-1) hexamethonium hydroxide electrolyte neutralized with triethanolamine to pH 8.0, using indirect UV detection at 254 nm. However, the detection sensitivity for tetrathionate was poor. Other polythionates can not be detected at all because of their high absorbance even at 254 nm. The developed CE method was applied for the monitoring of sulfur species in spent fixing solutions during the electrolytic oxidation.