I.V. Berezin

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.

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.

The principles of enzyme stabilization. III. The effect of the length of intra-molecular cross-linkages on thermostability of enzymes.

Abstract The effect of intramolecular cross-linkages of different length on the thermostability of α-chymotrypsin has been studied. To this end, the carboxy groups of the enzyme were first activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; then the activated proteins was treated by diamines of NH2(CH2)n NH2 type with n ranging from 0 to 12. The dependence of the rate constant of monomolecular thermoinactivation of the enzyme on the length of the cross-linking agent gives a minimum corresponding to tetramethylenediamine. The number of cross-linkages may be increased by enriching the protein molecule with carboxy groups; for this purpose α-chymotrypsin was succinylated. For succinylated α-chymotrypsin, the dependence of the rate constant of monomolecular thermoinactivation of the enzyme on the length of the cross-linking agent has a minimum for a shorter bifunctional reagent, ethylenediamine. In addition, the maximum stabilizing effect (compared to the native enzyme) increases (from 3- to 21-fold) of instead of α-chymotrypsin modified with tetramethylenediamine, succinylated α-chymotrypsin modified with ethylenediamine is used. This approach may be generally employed for preparation of stabilized water-soluble enzymes.

The principles of enzyme stabilization IV. Modification of ‘key’ functional groups in the tertiary structure of proteins

The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.

Determination of the individual rate constants of α‐chymotrypsin‐catalyzed hydrolysis with the added nucleophilic agent, 1,4‐butanediol

Knowledge of intermediary stage rate constants of enzyme reactions provides very valuable information about the mechanism both of separate reaction stages and of a process as a whole. Despite this, publisheddata on values of rate constants of ‘elementary’ reactions even for such an amply described enzyme as o-chymotrypsin are very scanty. To some extent, this is due to the lack of a sufficiently simple and reliable method of determining the constants. Hydrolysis of esters in the presence of cr-chymotrypsin proceeds according to a mechanism involving at least three steps [l] :

MECHANISM OF H2-ELECTROOXIDATION WITH IMMOBILIZED HYDROGENASE

Abstract The reaction of hydrogen electrooxidation on carbon-black electrodes with hydrogenase has been studied. It is shown that in the presence of hydrogenase a hydrogen equilibrium potential equal to 0.0 V is established on a carbon-black electrode in phosphate buffer, pH 7.5, saturated with hydrogen. The kinetic parameters have been studied and the mechanism of the hydrogen electroenzymic oxidation reaction has been determined.

Chemical modification of the ε-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.

The Kinetic Theory and the Mechanisms of Micellar Effects on Chemical Reactions

A few years ago we suggested, as an explanation of micellar effects on chemical reactions, a comprehensive kinetic theory which takes into consideration a partition of the reagents between the bulk and micellar “phases”, the simultaneous course of the reaction in the two phases and the shift of the apparent ionization constant of one of the reagents under the action of the surface micelle charge. In terms of this theory, from “surfactant concentration versus overall rate” profiles one can obtain partition coefficients of reagents between the bulk and micellar phases and a true rate constant of the reaction going in a micellar medium. That the kinetic equations are true is confirmed by the fact that the partition coefficients obtained in this way are in conformity with the values obtained by other methods (gel filtration, solubilization, spectrophotometric titration, etc.).

Enzymes Entrapped Into Reversed Micelles Of Surfactants In Organic Solvents: Key Trends In Applied Enzymology (Biotechnology)

This paper discusses applications of enzymes solubilized by surfactants in organic solvents to fine organic syntheses, in clinical and chemical analysis, and in therapy, as well as some future trends in biotechnology.