High Permeability of Polyunsaturated Lipid Bilayers As Applied to Attoliter Enzyme Reactors

Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes is their low reactivity because of the permeability resistance of lipid bilayers to substrates. In the present work, the polyunsaturated bilayers of 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3-PC) were elucidated to be highly permeable to 5(6)-carboxyfluorescein at high temperatures up to 60 °C and applied to fabricate vesicle-based reactive enzyme reactors. d-Amino acid oxidase (DAO) from porcine kidney was encapsulated in 18:3-PC vesicles with each aqueous volume of 3.4 × 10^-21 m³ (=3.4 aL). The DAO-containing vesicles were highly reactive at 40 °C toward d-alanine being added to bulk solution at pH 9.0 and stably catalyzed following two types of reactions. One is the DAO-catalyzed continuous production of H₂O₂ in the vesicles for 30 min being detected by the free peroxidase-catalyzed oxidation of o-dianisidine in bulk solution. The other is the cascade reaction in the vesicles coencapsulating DAO and catalase being followed for 5 h on the basis of the concentration of unreacted d-alanine. In the latter reaction, the intermediate product H₂O₂ was decomposed by catalase producing oxygen allowing its cyclical use for the DAO-catalyzed oxidation. Furthermore, thanks to the highly temperature-dependent permeability of 18:3-PC bilayers, on/off-like switching in the activity could be induced with respect to the vesicle-confined enzyme by shifting the reaction temperature between 20 and 40 °C. The above reactive vesicles can offer the opportunity of the H₂O₂-based reliable detection of d-amino acids and the continuous optical resolution of racemic mixtures of amino acids.