Kinetics of enzyme-catalysed de-symmetrisation of prochiral substrates : product enantiomeric excess is not always constant

The kinetics of enzymatic desymmetrisation were analysed for the most common kinetic mechanisms: ternary complex ordered (prochiral ketone reduction); ping-pong second (ketone amination, diol esterification, desymmetrisation in the second half reaction); ping-pong first (diol ester hydrolysis) and ping-pong both (prochiral diacids). For plausible values of enzyme kinetic parameters, the product enantiomeric excess (ee) can decline substantially as the reaction proceeds to high conversion. For example, an ee of 0.95 at the start of the reaction can decline to less than 0.5 at 95% of equilibrium conversion, but for different enzyme properties it will remain almost unchanged. For most mechanisms a single function of multiple enzyme rate constants (which can be termed ee decline parameter, eeDP) accounts for the major effect on the tendency for the ee to decline. For some mechanisms, the concentrations or ratios of the starting materials have an important influence on the fall in ee. For the application of enzymatic desymmetrisation it is important to study if and how the product ee declines at high conversion

Thermodynamic favorability of end products of anaerobic glucose metabolism

The eQuilibrator component contribution method allows calculation of the overall Gibbs energy changes for conversion of glucose to a wide range of final products in the absence of other oxidants. Values are presented for all possible combinations of products with up to three carbons and selected others. The most negative Gibbs energy change is for the formation of graphite and water (-499 kJ mol-1) followed by CH4 and CO2 (-430 kJ mol-1), the observed final products of anaerobic digestion. Other favored products (with various combinations having Gibbs energy changes between-300 and-367 kJ mol-1) are short-chain alkanes, fatty acids, dicarboxylic acids, and even hexane and benzene. The most familiar products, lactate and ethanol + CO2, are less favored (Gibbs energy changes of-206 and-265 kJ mol-1 respectively). The values presented offer an interesting perspective on observed metabolism and its evolutionary origins as well as on cells engineered for biotechnological purposes

Enzyme hydrolysis of cassava peels : treatment by amylolytic and cellulolytic enzymes

Cassava peels provide a cheap non-food biomass waste that can be hydrolyzed to simple sugars as a useful feedstock. Unlike most crop wastes, they have high starch content as well as lignocellulose. In this study, an enzymatic treatment of cassava peels by various concentrations of amylase and glucoamylase is considered. Steam explosion pre-treatments reduced rate and yield of hydrolysis. Milled peels suspended at 10% w/v yielded a maximum reducing sugar of 0.41 g (as glucose) per gram of peels. HPLC analysis showed that levels of soluble oligosaccharides remained low throughout. A pretreatment with amylase at 95 °C slightly increased rates although final yield was the same. Additional treatment with cellulolytic enzymes increases the total hydrolysis yield to 0.61 g (as glucose) per gram of peels representing 91% of the carbohydrate in cassava peels

Extensive counter-ion interactions seen at the surface of subtilisin in an aqueous medium

The extent of protein and counter-ion interactions in solution is still far from being fully described and understood. In low dielectric media there is documented evidence that counter-ions do bind and affect enzymatic activity. However, published crystal structures of macromolecules of biological interest in aqueous solution often do not report the presence of any counter-ions on the surface. The extent of counter-ion interactions within subtilisin in an aqueous medium has been investigated crystallographically using CsCl soak and X-ray wavelength optimised anomalous diffraction at the Cs K-edge. Ten Cs+, as well as six Cl- sites, have been clearly identified, revealing that in aqueous salt solutions ions can bind at defined points around the protein surface. The counter-ions do not generally interact with formal charges on the protein; formally neutral oxygens, mostly backbone carbonyls, mostly coordinate the Cs+ ions. The Cl- ion sites are also found likely to be near positive charges on the protein surface. The presence of counter-ions substantially changes the protein surface electrical charge. The surface charge distribution on a protein is commonly discussed in relation to enzyme function. The correct identification of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function

Supramolecular fibers in gels can be at thermodynamic equilibrium : a simple packing model reveals preferential fibril formation versus crystallization

Low molecular weight gelators are able to form nanostructures, typically fibers, which entangle to form gel-phase materials. These materials have wide-ranging applications in biomedicine and nanotechnology. While it is known that supramolecular gels often represent metastable structures due to the restricted molecular dynamics in the gel state, the thermodynamic nature of the nanofibrous structure is not well understood. Clearly, 3D extended structures will be able to form more interactions than 1D structures. However, self-assembling molecules are typically amphiphilic, thus giving rise to a combination of solvophobic and solvophilic moieties where a level of solvent exposure at the nanostructure surface is favorable. In this study, we introduce a simple packing model, based on prisms with faces of different nature (solvophobic and solvophilic) and variable interaction parameters, to represent amphiphile self-assembly. This model demonstrates that by tuning shape and "self" or "solvent" interaction parameters either the 1D fiber or 3D crystal may represent the thermodynamic minimum. The model depends on parameters that relate to features of experimentally known systems: The number of faces exposed to the solvent or buried in the fiber; the overall shape of the prism; and the free energy penalties associated with the interactions can be adjusted to match their chemical nature. The model is applied to describe the pH-dependent gelation/precipitation of well-known gelator Fmoc-FF. We conclude that, despite the fact that most experimentally produced gels probably represent metastable states, one-dimensional fibers can represent thermodynamic equilibrium. This conclusion has critical implications for the theoretical treatment of gels

Estimation of flattening coefficient for absorption and circular dichroism using simulation

The absorbance and circular dichroism (CD) of suspensions is lower than if the same amount of chromophore were uniformly distributed throughout the medium. Several mathematical treatments of this absorption flattening phenomenon have been presented using various assumptions and approximations. This article demonstrates an alternative simulation approach that allows relaxation of assumptions. On current desktop computers, the algorithm runs quickly with enough particles and light paths considered to get answers that are usually accurate to better than 3%. Results from the simulation agree with the most popular analytical model for 0.01 volume fraction of particles, showing that the extent of flattening depends mainly on the absorbance through a particle diameter. Unlike previous models, the simulation can show that flattening is significantly lower when volume fraction increases to 0.1 but is higher when the particles have a size distribution. The simulation can predict the slope of the nearly linear relationship between flattening of CD and the absorbance of the suspension. This provides a method to correct experimental CD data where volume fraction and particle size are known

Two-dimensional 1H and 1H-detected NMR study of a heterogeneous biocatalyst using fast MAS at high magnetic fields

Abstract Nuclear magnetic resonance (NMR) is a powerful tool for investigating atomic-scale structure in heterogeneous or composite materials where long-range order is absent. In this work solid-state 1H and 1H-detected NMR experiments were performed with fast magic angle spinning (νR = 75 kHz) and at high magnetic fields (B0 = 20 T) and used to gain structural insight into a heterogeneous biocatalyst consisting of an enzyme, human carbonic anhydrase II (hCA II), covalently immobilized on epoxy-functionalized silica. Two-dimensional 1H-1H NOESY-type correlation experiments were able to provide information on 1H environments in silica, epoxy-silica and the immobilized enzyme. Two distinct signals originating from water protons were observed: water associated with the surface of the silica and the water associated with the immobilized enzyme. Additional two-dimensional 1H-1H double–single quantum (DQ-SQ) correlation experiments suggested that the immobilized enzyme is not in close contact with the silica surface. Most significantly, comparison of two-dimensional 1H-15N spectra of the immobilized enzyme and the solution-state enzyme confirmed that the structural integrity of the protein is well preserved upon covalent immobilization

High-resolution structural characterization of a heterogeneous biocatalyst using solid-state NMR

Solid-state magic-angle spinning (MAS) NMR spectroscopy was employed to investigate structural detail in an enzyme, hu- man carbonic anhydrase II (hCA II) in uniformly 15N and selectively (15N leucine) enriched states, covalently immobilized on epoxy- functionalized silica. The immobilized hCA II retained 71% of its specific enzymatic activity when compared to the free enzyme in solution. Based on the one- and two-dimensional 1H, 13C, 15N and 29Si MAS NMR spectra, chemical shift assignments could be obtained from the silica support, covalent linker and the immobilized enzyme. The successful covalent immobilization of the enzyme on epoxy-silica was confirmed by the appearance of signals from the aromatic and carbonyl groups in the immobilized enzyme in addition to signals from the modified sup- port. Most notably, our MAS NMR results suggest that the covalent immobilization of the hCA II on epoxy-silica does not significantly affect the structural integrity of the protein

Modeling the kinetics of enzymic reactions in mainly solid reaction mixtures

There is currently considerable interest in using mainly solid reaction mixtures for enzymic catalysis. In these reactions starting materials dissolve into, and product materials crystalize out of, a small amount of liquid phase in which the catalytic reaction occurs. An initial mathematical model for mass transfer effects in such systems is constructed using some physically reasonable approximations. The model equations are solved numerically to determine how the reactant concentrations vary with time and position. To evaluate the extent to which mass transfer limits the overall rate of product formation, an effectiveness factor is defined as the ratio of the observed total reaction rate to the total reaction rate in the reaction limited limit. As expected, the value of the effectiveness factor in steady state is strongly dependent on the Thiele modulus. However, it is also observed that the effectiveness factor can vary widely as a result of changes in the other dimensionless groups characterizing the system. For example, there are situations with Thiele modulus equal to unity in which the value of the effectiveness factor varies between approximately 0.1 and 0.8 as the other parameters are varied in physically reasonable ranges. Analytical asymptotic solutions that provide good approximations to the numerically calculated results in various physically important limiting cases are also presented

A fluid-dynamical model for ‘anti-surfactants’

We construct a fluid-dynamical model for the flow of a solution with a free surface at which surface tension acts. This model can describe both classical surfactants, which decrease the surface tension of the solution relative to that of the pure solvent, and ‘anti-surfactants’ (such as many salts when added to water, and small amounts of water when added to alcohol) which increase it. We demonstrate the utility of the model by considering the linear stability of an infinitely deep layer of initially quiescent fluid. In particular, we predict the occurrence of a novel instability driven by surface-tension gradients, which occurs for anti-surfactant, but not for surfactant, solutions