Karel Martinek

Engineering biocatalytic systems in organic media with low water content

Abstract The use of organic media in biocatalysis stems from the fact that in many cases biocatalytic processes can hardly be conducted (if at all) in aqueous solutions because of extremely low solubilities of substrates and/or unfavorable shift of the reaction equilibrium in water. The growing interest in this biotechnological area that has sprung up over the past few years has resulted in various approaches to enzyme stabilization against organic solvents. Thus, the main goal of the present review is to formulate a comprehensive classification of numerous successful nonaqueous biocatalytic systems based on a few fundamental principles. Typical examples are considered, along with the advantages and drawbacks inherent in each of the approaches discussed.

Micellar enzymology: its relation to membranology

Micellar enzymology, a new trend in molecular biology, studies catalysis by enzymes entrapped in hydrated reversed micelles composed of surfactants (phospholipids, detergents) in organic solvents. The key research problems of micellar enzymology and its relation to enzyme membranology are discussed.

The principles of enzyme stabilization I. Increase in thermostability of enzymes covalently bound to a complementary surface of a polymer support in a multipoint fashion

The general principle of enzyme stabilization has been formulated: inactivation of the enzyme due to unfolding of its molecule under a certain denaturing action may be sharply retarded provided the protein globule is rigidified by being attached to a complementary surface of a relatively rigid support in a multipoint fashion. A method has been elaborated allowing a support with a surface geometry strictly congruent to that of the enzyme globule to be prepared and ensuring multipoint covalent binding to be effected. To this end, the enzyme molecule is modified in many points by a monomer analogue and the resulting enzyme preparation is copolymerized with the monomers. As a result, the enzyme proves to be attached with multiple linkages in the three-dimensional lattice of polymeric gel. The method of enzyme stabilization has been subjected to experimental verification. Model enzymes, chymotrypsin and trypsin, were first acylated by acryloyl chloride or coupled with acrolein then copolymerized with the monomers, sodium methacrylate or acrylamide. The thermostability of the immobilized enzymes obtained as a result is by several orders of magnitude higher (103−108 times above the 60–102°C temperature range) than that of native enzymes, with high catalytic activity being retained. This technique allows preparation of both highly stable water-insoluble enzymes (on formation of gel cross-linked with N,N′-methylene bisacrylamide) and soluble stabilized enzymes (if polymerization is performed without other than the enzyme cross-linking agents).

The principles of enzymes stabilization: VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents☆

1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

Structure-stability relationships in proteins: new approaches to stabilizing enzymes

Abstract A comparison of the structure of (a) proteins from thermophilic and mesophilic microorganisms, (b) closely related proteins with different thermostability from various mesophilic sources, and (c) mutationally altered enzymes with those from wild strains has been carried out. The main molecular mechanisms existing in nature for the creation of thermostable proteins have been elucidated. The most important mechanism is the strengthening of hydrophobic interactions in the interior of the protein globule. This mechanism has been employed to advance a novel approach to enzyme stabilization which consists of the following steps. A protein is first made to unfold into a random coil-like state and then the folding of the protein is performed in one of three ways: (1) in ‘non-native’ conditions, (2) in the presence of substances which can interact with the protein in a noncovalent fashion, (3) after covalent modification of the unfolded protein.

The principles of enzyme stabilization. II. Increase in the thermostability of enzymes as a result of multipoint noncovalent interaction with a polymeric support.

Abstract The catalytic activity, thermostability (resistance to monomolecular thermo-inactivation) and molecular mobility of chymotrypsin and trypsin mechanically entrapped into polymethacrylate and polyacrylamide gels have been studied. It has been established that the thermostability of the enzymes does not depend on the concentration of electroneutral polyacrylamide gel over the range of 0–50 w/w%. However, in polymethacrylate gel of concentration higher than 30 w/w%, when a high catalytic activity is retained, the thermostability of chymotrypsin dramatically increases: in 50 w/w% gel the first-order rate constant for thermoinactivation of the enzyme at 60°C is 10−5 that in water. Based on these data and also on experimentally obtained results on transitional and rotational diffusion of both native and modified enzymes, the following mechanism of enzyme stabilization is formulated and proved. In principle, the protein molecule of an enzyme may form with the three-dimensional lattice of polyelectrolyte gel multiple noncovalent linkages (via electrostatic or hydrogen bonds); as a result, the structure of the enzyme becomes more rigid and its thermostability should increase. However, since these bonds are relatively weak, in diluted gels they can hardly be realized, as the “quenching” of the transitional movement of the enzyme molecules, accompanying complex formation would have required a heavy entropy loss. At the same time, in concentrated gels, this unfavourable entropy contribution is absent as the polymers lattice provides significant steric hindrances for the transitional diffusion, so that the molecules almost stop moving. That is why weak linkages between the protein globule and the support can be realized here. That the complex formation does take place is indicated by the fact the rotational diffusion of the protein molecules is almost completely frozen. When there is no specific protein-support interaction (in polyacrylamide gel), no deceleration of the rotational movement of the protein molecules occurs and no noticeable increase in the thermostability of the enzymes is observed. It is possible that the mechanism discovered by us functions in vivo and is responsible for the stability (and, which is important, for stability regulation) of the proteins incorporated in biomembranes. On the other hand, the results obtained by us may enrich enzyme engineering, as they allow the general strategy of production of stabilized enzymes to be outlined.

Enzymes entrapped into reversed micelles in organic solvents: Sedimentation analysis of the protein—aerosol OT-H2O-Octane system

Abstract Ultracentrifugation was used to study the systems of reversed Aerosol OT micelles in octane that contain solubilized protein (α-chymotrypsin, lysozyme, trypsin, egg albumin, horse liver alcohol dehydrogenase, γ-globulin). Changes in the sedimentation coefficients of reversed micelles upon protein entrapment into the latter were found to correlate solely with the molecular weight of solubilized protein in a wide range of experimental conditions, such as the surfactant hydration degree or protein concentration. Proceeding from this, a simple model of solubilization was suggested according to which a protein molecule is entrapped into a reversed micelle in a stoichiometric ratio of 1:1 rendering therewith no significant effect on the size of the reversed micelle. The conditions were found by the example of α-chymotrypsin under which the sedimentation properties of the system deviate from those of the model. The deviations occur at rather low hydration degrees of the surfactant when the inner cavity of a reversed micelle is less than the effective size of the solubilized protein molecule. In the latter cause the protein “creates” around itself a new micelle of a required (bigger) size.

Strategy for Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction with a Support

This review states that the covalent multi-point attachment of enzymes to a support is the most general approach to stabilize them against different denaturing conditions, namely against their inactivation caused by protein unfolding. It is suggested that the change in the wavelength of the maximum emission in fluorescence spectra of a protein, resulting from its denaturation, can be used to evaluate a priori the effectiveness of stabilization. The copolymerization method of enzyme immobilization, as the most promising approach to stabilizing enzymes, is discussed in detail.

Structure-stability relationship in proteins; fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Enzymes entrapped in reversed micelles of surfactants in organic solvents: A theoretical treatment of the catalytic activity regulation

General regularities of catalysis by enzymes solubilized in reversed micelles of surfactants in organic solvents are discussed. The kinetic scheme describing the observed dependency of catalytic activity on surfactant hydration and concentration is presented, and a computer simulation is performed of the theoretical equations. Finally, possible mechanisms and the range of enzyme activity regulation in reversed micellar systems are qualitatively analysed.