Investigation of behavior of an enzyme in a biphasic system: soybean lipoxygenase-1

Soybean lipoxygenase-1 (EC 1.13.11.12) reaction with linoleic acid as substrate was used to study the biocatalysis in a biphasic system when the reactants have surface-active properties. The poorly water-soluble substrate was initially dissolved in an apolar solvent (octane). The hydroperoxide produced was water soluble and remained in the aqueous phase (borate buffer). The bioreactor was a modified Lewis cell with a well-defined interfacial area between the two phases. Two phenomena were studied separately: the reactant transfer between the two phases and the biocatalyzed reaction in an aqueous medium. This allowed determination of the transfer and the reaction constants. Substrate transfer was found to be affected by the progress of the reaction, because linoleic acid and the hydroperoxy acid have an influence on the interfacial tension. Inactivation of the biocatalyst at the interface was observed in the bioreactor. These results indicate that it is impossible to analyze the system behavior with the method proposed in the literature, which is based on the sequential study of the substrate transfer to the aqueous phase and its biocatalysis by lipoxygenase. The interaction between transfer phenomena and reaction kinetics was studied in the biphasic system. The kinetics were different from those obtained in the aqueous medium. Catalysis and transfer influence each other reciprocally. In this compartmentalized system, cooperativity phenomena were obtained using a nonallosteric enzyme. The evolution of the system was modeled (Runge-Kutta algorithm). The curves obtained were very close to those determined experimentally.