Thermodynamic predictions for biocatalysis in nonconventional media: theory, tests, and recommendations for experimental design and analysis.

Tetrahedron report number 203

Enzyme stabilization by immobilization

Engineering biocatalytic systems in organic media with low water content

The potentials of using organic reaction media in biotechnological conversions have already been demonstrated in several experimental studies. Examples of possible advantages are: possibility of higher substrate and/or product concentrations, favorable shift of reaction equilibria, reduced substrate and/or product inhibition, and facilitated product recovery. Especially water/organic solvent two-phase systems seem to possess several of these advantages. The solvent type will highly affect kinetics and stability of the (immobilized) biocatalyst, solubility and partitioning of reactants/products, and product recovery. Therefore the solvent choice can have a large influence on the economics of the two-liquid-phase biocatalytic process. Immobilization of the biocatalyst may be useful to provide protection against denaturating solvent effects. The polarity of the employed support material will also be decisive for the partitioning of substrates and products among the various phases. A classification of biphasic systems, which is based on the possible types of theoretical concentration profiles and aqueous phase configurations, is discussed. Reversed micelles and aqueous two-liquid-phase systems can be considered as special cases. The design of two-liquid-phase bioreactors is dependent on the state of the biocatalyst, free or immobilized, and on the necessity for emulsification of one of the two liquid phases in the other. Many mass-transfer resistances, e.g. across the liquid/liquid interface, in the aqueous phase, across the liquid/solid interface, and in the biocatalyst phase, can limit the overall reaction rate. The epoxidation of alkenes in water/solvent two-phase systems is discussed to give an example of the scope of biotechnological processes that is obtained by using organic media. Finally, a design calculation of a packed-bed organic-liquid-phasel immobilized-biocatalyst reactor for the epoxidation of propene is given to illustrate some of the above aspects.

Biocatalysis in Organic Media

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the systhesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

Gennady P. Samokhin

Papers

A new approach to preparative enzymatic synthesis.