Jean-Marc Engasser

Effect of internal diffusion in heterogeneous enzyme systems: evaluation of true kinetic parameters and substrate diffusivity.

Abstract The effect of internal diffusion on the overall reaction rate in spherical particles and membranes containing immobilized enzymes has been investigated theoretically. Since they represent open systems, the MichaelisMenten kinetics is obeyed in the absence of diffusional effects at steady state even at high enzyme concentrations. When internal diffusion perturbs the reaction, the system can not be described any more by KM and Vmax‴ alone, but is conveniently characterized by the modulus. Assuming that only internal diffusion interferes with the enzyme reaction, the effect of the modulus on the overall rate of reaction is illustrated by the results of computer calculations. Plots of the overall reaction rate against the substrate concentration are hyperbolas at various moduli for both membranes and spherical particles and no sigmoidal curves are obtained with immobilized enzyme systems. Since the conventional plots of enzyme kinetics do not yield straight lines under such conditions, a graphical method is proposed to determine KM and Vmax‴ as well as the substrate diffusivity in the enzymic medium.

Diffusion and Kinetics with Immobilized Enzymes1

Publisher Summary This chapter discusses the analysis of the kinetic behavior of enzymes entrapped in or bound to membranes or other supports, as can be observed from macroscopic measurements. The growing employment of immobilized enzymes in various technological applications requires an understanding of the overall kinetic properties to design the systems in which the potential of enzymes can be fully exploited. Immobilized enzymes can also serve as experimental and theoretical models for bound enzymes in living systems so that their study has a broader scope than that of technological utility. It is recognized that in the cellular environment, most enzymes are localized in various cell compartments and the catalytic properties can be different from those of the same enzymes in free solution. The phenomena related to the structure of the heterogeneous enzymic environment are receiving more attention in cellular physiology. Soil chemistry and other areas of agricultural chemistry also provide a theater of great scientific and practical significance for the action of bound enzymes.

Buffer-facilitated proton transport. pH profile of bound enzymes

Abstract 1. 1. In heterogeneous systems conjugate acid base pairs, besides their conventionally accepted static role as buffers, can also play a dynamic role in facilitating proton transport. When protons are generated and consumed at different locations, the conjugate base binds the proton at the “source” and the resulting acid diffuses to the “sink”. 2. 2. Both the buffering capacity of an acid base pair and its ability to facilitate proton transport are maximum when its p K A is close to the pH of the medium. The transport facilitating effect can be appreciable at much lower concentrations than those needed for buffering capacity. 3. 3. The effect of proton transport has been theoretically investigated by using a model consisting of a H+-producing enzymic surface reaction, which occur with bound enzymes both in vitro and in vivo. 4. 4. The enhancement of proton transport is quantitatively expressed by the extent of transport facilitation, ξ. The interplay of facilitated proton diffusion and reaction kinetics is quantified by the proton modulus, μ. 5. 5. At a given p K A the magnitude of transport facilitation increases with the acid-base concentration and the pH of the solution. The p K A value plays a dual role in determining both the concentration of available carriers and the affinity of the proton to the carrier. 6. 6. Due to its high proton affinity OH− can also facilitate proton transport already at pH 7, but in the presence of buffers its effect becomes significant only at higher pH values. 7. 7. When the extent of transport facilitation sharply increases with the pH of the substrate solution, at relatively low buffer concentrations bound enzymes display sigmoidal pH profiles with sharp peaks. Previously reported experimental data with membrane bound enzymes can be interpreted in view of these theoretical findings. 8. 8. It is postulated that buffers ubiquitous in biochemical and living systems can play an important role also by facilitating proton transport.

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Abstract Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l−1), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l−1 h−1) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.

Comparative study of some surface active properties of fructose esters and commercial sucrose esters

Abstract Surface active properties (i.e. oil–water interfacial tension γ o/w , air–water surface tension γ a/w , critical micelle concentration (CMC), foaming and emulsion stability) of different enzymatically synthesized pure monoesters and blends of mono- and difructose esters were investigated and compared with those of some sucrose esters (P1670, S1670, SP30, SP70). The results show that fructose esters exhibited interesting surface properties. Indeed, fructose esters with low chain lengths led to the lowest values of surface tension whereas for high chain lengths of fatty acids, γ a/w values were similar to those obtained with sucrose esters. However, fructose esters exhibited higher interfacial tension values compared with commercial sucrose esters. The results also show that depending on the carbon chain length and the monoester/diester ratio, fructose esters may be more advantageous than sucrose esters. Actually, the highest oil–water emulsion stability was reached with pure fructose monocaprate and blend of mono- and dicaprate. Moreover, the best initial foaming power was reached with fructose monocaprate and was comparable to that of P1670 after 45 min.

Comparison of intrinsic stabilities of free and bound enzymes by graphical removal of diffusional effects

The enhanced stability usually exhibited by enzymes after immobilization may be attributed either to a stabilization effect of the solid matrix on the bound enzyme molecule or to the influences of diffusional limitations on the observed activity. To allow the comparison of the intrinsic statilities of free and bound enzymes a simple graphical procedure for the removal of external diffusional effects of stability curves is described. It is based on the determination of substrate concentration differences between the enzyme micro- and macroenvironment. Application of the method to aspartate aminotransferase bound to collagen membranes indicates that diffusional limitations for oxaloacetate are partly responsible for the observed stability enhancement. Comparison of the graphically obtained intrinsic profile with the stability curve of the soluble enzyme further demonstrate that the binding itself greatly increases the stability of aspartate aminotransferase.

A simple additivity relation for analysis of heterogeneous catalytic reactors with first order reaction

An isothermal heterogeneous reactor with first order kinetics is characterized by two virtual maximum efficiencies, Nkin and Ntr, which are readily determined from the appropriate kinetic and transport parameters. The actual efficiency of the reactor, which is related to the conversion, X, by X = 1−e−N, can be accurately approximated as N−1 = N−1tr + N−1kin. The overall additivity relation offers a simple approach either to predict the conversion or to extract the kinetic or transport parameters when the reactor behavior is affected by both reaction and diffusion.

Facilitated diffusion with consecutive reaction: Optimal carrier affinity☆

Abstract The interplay between facilitated diffusion of a substrate through a membrane and a consecutive enzymic reaction, both of which follow Michaelis-Menten kinetics, has been theoretically investigated and the effect of the kinetic and transport parameters on the rate of substrate uptake is graphically illustrated. At steady state two characteristic features of the system have been identified. First, the substrate concentration at the internal enzymic side of the membrane cannot exceed a given value even at much higher external substrate concentrations. Second, the uptake rate is maximum at a given value of K T , the kinetic parameter of the transport system that expresses the reciprocal carrier affinity of the substrate. The optimum value of K T is approximately equal to the external substrate concentration. This particular dependence of the uptake rate on the carrier affinity is expected to play an important role in hormonal regulation.