Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Enzymatic reaction kinetic: comparison in an organic solvent and in supercritical carbon dioxide

Myristic acid esterification has been performed by an immobilized lipase from Mucor Miehei both in n-hexane and in supercritical carbon dioxide (SCCO(2)). The enzyme is stable in SCCO(2) at 15 MPa and 323 K. The reaction rate is influenced by the concentration of water and by the reaction medium composition. A reaction mechanism is proposed, and kinetic parameters are determined at 12.5 MPa and 313 K. Maxium velocity appears 1.5-fold higher in SCCO(2) than in n-hexane; however, as solubility of myristic acid is greater in n-hexane, it is not yet definitively clear that the supercritical medium is more favorable than the classical organic solvent for this type of enzyme reaction.     

Enzyme stability in the presence of organic solvents

Summary The thermal stability of vacuum-dried acid-phosphatase has been investigated, both in the absence and in the presence of pure hexadecane. Preliminary experimental results indicate that: i) in both solid-phase runs, acid-phosphatase is much more stable than the free enzyme in aqueous solution, ii) the presence of the organic solvent slightly reduces thermal stability of the solid-phase enzyme. As regards the deactivation mechanism, when acid-phosphatase operates in free aqueous solution it follows a two-step in series deactivation. Initially the native configuration decays towards an intermediate, still active form. This, in turn, irreversibily yields a totally inactive structure. In the thermal deactivation of solid-phase enzyme it has been observed that: i) the first step is substantially retarded, ii) the final transition is completely hindered, iii) the intermediate configuration is more active than that produced in aqueous solution, by more than one order of magnitude.     

Factors affecting immigration of adults: experimental and theoretical observations with rodents

We examined immigration in populations of Peromyscus leucopus (white-footed mice) by 1) monitoring natural immigration at 10 sites, 2) introducing experimental immigrants into eight populations, and 3) constructing qualitative models of immigration. Density of natural immigrants covaried positively with resident density, and successful assimilation was lower at low resident and immigrant densities. Females and males did not differ in their chance of achieving residency. Year, sex, and number introduced were significant predictors of assimilation by individuals. Experimentally introduced individuals had no effect on densities of those mice resident before the introductions. Results obtained from studying natural immigration differed from those obtained from studying experimental immigration. Signed digraphs and time averaging were used to model the consequences of different resident-immigrant relationships and to suggest how different sources of variation may have affected assimilation and led to differences in natural and experimental immigration. Resident and immigrant densities could have been positively correlated even if residents actively inhibited immigration. Variation in immigrant density and survival rather than resident territorial activity apparently determined patterns of assimilation.     

A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum

A theoretical model of bubble dissolution in xylem conduits of stems was designed using the finite differential method and iterative calculations via computer. The model was based on Fick's, Henry's and Charles' laws and the capillary equation. The model predicted the tempo of recovery from embolism in small diameter branches of woody plants with various xylem structures under different xylem water pressures. The model predicted the time required to recover conductivity in any position in the stem. Repeated iterative solution of the model for different situations yielded an empirical formula to calculate the time for complete recovery of conductivity in stems from a fully embolised initial state. The time, t_p, is given by: where α is a temperature coefficient; D is the coefficient of diffusion of air in wood at 25°C; r_cs is the ratio of the area of total conduit cross section to the stem cross section; Ψ_xp is the stem xylem pressure potential (Pa, where 0 Pa equals atmospheric pressure); τ is solution surface tension (0.072 N m^?1); and D_c and D_s are diameters of the conduits and the stem, respectively (m). The equation is valid only when Ψ_xp > –4τ/D_c. The model predicts no recovery of conductivity when Ψ_xp≤–4τ/D_c. The model agreed with experiments.     

Enzyme and organic solvents: horse liver alcohol dehydrogenase in non-ionic microemulsion: stability and activity

In a microemulsion made with Triton X-100, the stability of the enzymatic activity was higher than in ionic microemulsions. The stability increased with water content. The kinetic constants (Michaelis constant of NAD+ and maximum velocity) were close to those found in the previously studied microemulsions. The Michaelis constant of NAD+ expressed with respect to the buffer volume was higher than in water. The pH dependence of the kinetic constants in this microemulsion was determined. The activity determined by NAD+ reduction decreased with water content, whereas the redox activity determined via butanol oxidation coupled to retinal reduction was only slightly reduced.     

Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents

Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed. (c) 1995 John Wiley & Sons, Inc.     

Enzyme reaction rates and the stochastic theory of kinetics

The kinetics of an elementary reaction step are discussed from the viewpoint of the stochastic theory of chemical kinetics. The general form of the rate constant found in the stochastic approach is described, and compared with the expression from transition state theory. Whereas the stochastic theory predicts a rate enhancement in cases which are not adiabatic (in the quantum mechanical sense), transition state theory,which is essentially an adiabatic theory of reaction rates, does not permit inclusion of the effect. This effect can be expected to be of greater importance in cases of catalysis by structures, such as enzymes, containing large numbers of vibrational degrees of freedom (particularly low frequency ones) than in cases lacking such structures. The stochastic theory is more general than the transition state theory, the rate constant expression given by the latter being obtainable from the former when restrictive assumptions, including that of adiabaticity, are made. Interpretations of enzyme catalysis based on the transition state theory must thus be viewed as speculative.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.     

Effect of organic solvents on penicillin acylase-catalyzed reactions: interaction of organic solvents with enzymes

The effects of various organic solvents on penicillin acylase-catalyzed synthesis of β-lactam antibiotics (pivampicillin and ampicillin) have been investigated in water-solvent mixtures. The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent. In particular, the rate of enzyme catalysis was extremely low in the presence of ring-structured solvents and acids while enzyme activities were fully restored after removing the solvents. This indicates that interactions between the solvents and the enzyme are specific and reversible. To correlate the inhibitory effects of organic solvents with solvent properties the influence of solvent hydrophobicities and solvent activity on the rate of pivampicillin synthesis was examined. The reaction rate was found to decrease with increasing solvent hydrophobicities, and a better correlation was observed between the reaction rate and solvent activity. The effects of ionic strength on the synthesis of pivampicillin and ampicillin were also examined. The ionic strength dependence indicates that electrostatic interactions are involved in the binding of ionic compounds to the enzyme. On the basis of the active site structure of penicillin acylase, a possible mechanism for molecular interactions between the enzyme and organic solvents is suggested.     

Enzyme kinetics for a two-step enzymic reaction with comparable initial enzyme-substrate ratios

We extend the validity of the quasi-steady state assumption for a model double intermediate enzyme-substrate reaction to include the case where the ratio of initial enzyme to substrate concentration is not necessarily small. Simple analytical solutions are obtained when the reaction rates and the initial substrate concentration satisfy a certain condition. These analytical solutions compare favourably with numerical solutions of the full system of differential equations describing the reaction. Experimental methods are suggested which might permit the application of the quasi-steady state assumption to reactions where it may not have been obviously applicable before.     

A theoretical model of the knee and ACL: theory and experimental verification.

A three-dimensional mathematical model of the human knee joint was developed to examine the role of single ligaments, such as an anterior cruciate ligament (ACL) graft in ACL reconstruction, on joint motion and tissue forces. The model is linear and valid for small motions about an equilibrium position. The knee joint is modeled as two rigid bodies (the femur and the tibia) interconnected by deformable structures, including the ACL or ACL graft, the cartilage layer, and the remainder of the knee tissues (modeled as a single element). The model was demonstrated for the equilibrium condition of the knee in extension with an anterior tibial force, causing anterior drawer and hyperextension. The knee stiffness matrix for this condition was measured for a human right knee in vitro. Predicted model response was compared with experimental observations. Qualitative agreement was found between model and experiment, validating the model and its assumptions. The model was then used to predict the change in graft and cartilage forces and joint motion of the knee due to an increment of load in the normal joint both after ACL removal and with various altered states simulating ACL reconstructions. Results illustrate the interdependence between loads in the ACL graft, other knee structures, and contact force. Stiffer grafts and smaller maximum unloaded length of the ligament lead to higher graft and contact forces. Changes in cartilage stiffness alter load sharing between ACL graft and other joint tissues.