Colloidal solution of water in organic solvents: a microheterogeneous medium for enzymatic reactions.

To simulate in vitro the conditions under which enzymes act in vivo, enzyme molecules have been entrapped in hydrated reverse micelles of a surfactant in organic solvents. In this system the catalytic activity of one of the enzymes studied (peroxidase) became much higher than in water, and the specificity of the other enzyme (alcohol dehydrogenase) was dramatically altered.

The evolution of biotransformation technologies.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

Biocatalysis in nonaqueous solvents.

Biotransformation technologies have enjoyed a renewed interest from researchers and industry because of the progress made in the discovery and design of new, efficient biocatalysts for synthetic applications. Biocatalysis in nonaqueous media, which offers unique capabilities and thus plays a major role in biotransformation technologies, has made tremendous progress in recent years. On average, at least one paper dealing with biocatalysis in organic solvents is published every day. New, remarkable developments have taken place in several key areas of this exciting field during the past year.

Combinatorial biocatalysis: a natural approach to drug discovery.

Nature's most potent molecules are produced by enzyme-catalysed reactions, coupled with the natural selection of those products that possess optimal biological activity. Combinatorial biocatalysis harnesses the natural diversity of enzymatic reactions for the iterative synthesis of organic libraries. Iterative reactions can be performed using isolated enzymes or whole cells, in natural and unnatural environments, and on substrates in solution or on a solid phase. Combinatorial biocatalysis is a powerful addition to the expanding array of combinatorial methods for the generation and optimization of lead compounds in drug discovery and development.

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

The stability of alpha-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.

Phospholipase D-catalyzed transphosphatidylation in anhydrous organic solvents.

A new reaction system suitable for phospholipase D (PLD)-catalyzed transphosphatidylation of alcohols with phosphatidylcholine under anhydrous conditions is reported. The key innovation of the reaction system is a cation-exchange resin serving as a scavenger for choline that forms as a byproduct in the transphosphatidylation reaction. Due to the absence of water in this system, the reaction path dramatically shifts in favor of the target transphosphatidylated product, whereas the undesirable side hydrolysis of phosphatidylcholine is completely suppressed, in contrast to commonly used biphasic water-organic systems. In addition, a salt activation technique is successfully applied to increase the catalytic activity of PLD in this anhydrous system. The new reaction system is successfully used for transphosphatidylation of a wide range of primary, secondary, and aromatic alcohols catalyzed by PLD from Streptomyces sp.

Detergentless microemulsions as media for enzymatic reactions. Cholesterol oxidation catalyzed by cholesterol oxidase.

Catalytic activity and stability of cholesterol oxidase dissolved in ternary systems composed of n-hexane, isopropanol, and water were studied. The dependence of catalytic activity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first-order inactivation rate constant of cholesterol oxidase dissolved in n-hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ.mol-1, respectively. Preparative conversion of cholesterol to cholestenone catalyzed by cholesterol oxidase in n-hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.

The effect of water content and nature of organic solvent on enzyme activity in low-water media. A quantitative description.

A simple theoretical model was suggested to describe quantitatively the effect of water content and nature of organic solvents on catalytic behavior of enzymes suspended in low-water media. The model was based on a generally accepted notion that the destruction of the protein hydration shell is one of the main reasons for protein denaturation by organic solvents. The validity of the model was confirmed by the example of catalytic behavior of immobilized laccase suspended in water/organic mixtures of different compositions. In addition, the results were used to demonstrate that the effect of organic solvents and/or water content on catalytic behavior of enzymes in low-water media can be adequately assessed only in terms of the full kinetic description based on properly determined Vm and Km values.

Kinetic theory of enzymatic reactions in reversed micellar systems. Application of the pseudophase approach for partitioning substrates.

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving substrates capable of partitioning among all pseudophases of the micellar system i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system.

Multipoint attachment to a support protects enzyme from inactivation by organic solvents: alpha-Chymotrypsin in aqueous solutions of alcohols and diols.

Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.

Dissociation of lactate dehydrogenase in aqueous and reversed micellar solutions. A time-resolved polarized fluorescence study.

Dissociation behavior of lactate dehydrogenase from hog muscle, both in aqueous solution and reversed micelles of sodium bis(2-ethylhexyl sulfosuccinate) in octane was studied using time-resolved polarized fluorescence spectroscopy. It was found that, in aqueous solutions, the enzyme underwent partial dissociation with the formation of isolated subunits at enzyme concentrations below 8 nM. Dissociation of the enzyme also took place upon entrapment of lactate dehydrogenase into reversed micelles under conditions of low surfactant hydration, when micelles were not large enough to accommodate a whole protein molecule.

Enzyme inactivation and protection during entrapment in reversed micelles.

Lactate dehydrogenase (LDH) was found to lose rapidly its catalytic activity during the process of entrapment in hydrated reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane. It was demonstrated that this inactivation was caused by the surfactant which penetrated into the injected enzyme-containing aqueous bulk phase during the short time of mechanical stirring needed to convert the initial biphasic mixture into a monophasic reversed micellar solution. The unfavorable inactivation phenomenon could be efficiently eliminated by the addition of either NADH or pyruvate into the enzyme stock solution prior to its injection into AOT solution in octane. The catalytic activity of substrate-protected LDH in AOT reversed micelles increased with increasing water content of the micellar system, reaching the maximal level above wo = 30 when aqueous inner cores of reversed micelles grew large enough to allow unrestricted accommodation of the enzyme molecule. It is suggested that the employment of substrate-protected enzymes could represent a generally useful approach for producing highly efficient enzyme-containing reversed micellar reaction systems.

Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent.

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor.

Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. Alpha-chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/formamide solvents.

The dependence of the catalytic activities of alpha-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.