ABSTRACT
D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the d-forms of alanine (Ala) and serine (Ser). The permeability coefficient of d-Ala and d-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.
