Andrey V. Levashov

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 neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles: Micelles as microcontainers for drug targeting

It has been suggested to use surfactant micelles as microcontainers for increasing the efficiency of neuroleptic targeting from blood flow into the brain. The neuroleptic action of haloperidol, intraperitoneally injected into mice in micellar solution of non‐ionic block copolymer surfactant (pluronic P‐85) in water, increased several‐fold if compared with that observed for haloperidol aqueous solution. Incorporation of brain‐specific antibodies into haloperidol‐containing micelles resulted in additional drastic increase (more than by 2 orders of magnitude) in the drug effect.

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.

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.

Bioorganic synthesis in reverse micelles and related systems

Abstract Reverse micelles (or w/o microemulsions) have found wide applications in enzymology, protein chemistry and other areas assisting in a variety of biotransformations. Being considered as an individual ‘nanobioreactors’ these systems allow one to reveal or to add new properties to biocatalysts.

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: Negatively charged groups artificially introduced into α-chymotrypsin provide additional activation and stabilization effects

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

Relationship between surface hydrophilicity of a protein and its stability against denaturation by organic solvents

The stability of α‐chymotrypsin covalently modified with a strongly hydrophilic modifier, pyromellitic dianhydride, against solvent‐induced denaturation in water—organic solvent binary mixtures has been studied. It was found that the hydrophilization results in a strong stabilization of the enzyme against denaturation by organic solvents. The stabilizing effect is explained in terms of the enhanced ability of the hydrophilized enzyme to keep its hydration shell, which is indispensable for supporting the native protein conformation, from denaturing stripping by organic solvents

Preparation of conjugates of progesterone with bovine serum albumin in the reversed micellar medium

Progesterone-BSA (bovine serum albumin) conjugates which contain up to 47 steroid molecules linked to a BSA molecule have been prepared by the activated ester method, the conjugation step being carried out in reversed micellar solutions of sodium di(2-ethylhexyl) sulphosuccinate (AOT) in octane. The number of incorporated steroid molecules increases on passing to increased water/AOT ratios at the given activated steroid/BSA ratio. The results show that the reversed micellar medium would be useful for preparation of conjugates of hydrophobic steroids with proteins in respect to simplicity and ease in obtaining conjugates with high steroid/protein ratios.

Cell-free translation in reversed micelles.

Cell‐free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin‐2 by the wheat germ translation system solubilized in Brij 96 (oleyl‐poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell‐free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2‐ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2− ions necessary for the functioning of the translation apparatus.