Oligolamellar vesicles for covalent immobilization and stabilization of D-amino acid oxidase.

Oligolamellar phospholipid vesicles incorporated with d-amino acid oxidase from porcine kidney (OV-DAO) were prepared by encapsulating pre-formed enzyme-bound unilamellar vesicles (UV-DAO) with bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer of UV-DAO was composed of POPC, 30 mol% of cholesterol and 15 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (NGPE) that was responsible for covalent linking to D-amino acid oxidase (DAO). OV-DAO and UV-DAO showed the activity to catalyze the oxidation of D-alanine as measured based on the hydrogen peroxide produced. The oligolamellar and unilamellar structure of OV-DAO and UV-DAO, respectively was elucidated based on the quenching characteristics of bilayers-incorporated fluorescent lipid 7-nitro-2,1,3-benzoxadiazol-4-yl-phosphoethanolamine (NBD-PE) and the size distribution of the vesicles measured with the dynamic light scattering method. The enzyme activity of OV-DAO and UV-DAO was significantly stabilized at 50°C compared to that of free DAO at the fixed enzyme concentration of 3.29 µg/mL. At the temperature, OV-DAO and UV-DAO showed the remaining activity of 52.7 and 29.6%, respectively at the incubation time of 20 min while free DAO was completely deactivated. Thus the dimeric form of DAO could be stabilized by its coupling to the surface of UV-DAO membrane being the inner bilayer of OV-DAO. Furthermore, the thermal denaturation of DAO and dissociation of flavin adenine dinucleotide (FAD) from the subunits of enzyme were prevented in the aqueous phase formed between the bilayers of OV-DAO.

Catalase-conjugated liposomes encapsulating glucose oxidase for controlled oxidation of glucose with decomposition of hydrogen peroxide produced.

The catalase-conjugated liposome encapsulating glucose oxidase (CLG) was prepared for developing a novel liposomal system for glucose oxidation with controllable enzyme activities. The catalase molecules were conjugated to the surface of liposome with 100 nm in mean diameter through coupling with the membrane-incorporated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (NGPE) at its mole fraction f(G) of 0.05 or 0.15. The average number of enzyme molecules per CLG with f(G) of 0.15 was 8.7 for glucose oxidase and 6.5 for catalase. The CLG-catalyzed oxidation of glucose was performed at 40 degrees C for prolonged period up to 99 h. The CLG with f(G) of 0.15 gave larger oxidation rate than that with f(G) of 0.05. In the fed-batch oxidation of glucose catalyzed by the former CLG, the stable oxidation rate was observed for 75 h with negligible accumulation of H(2)O(2) produced because of the durable catalytic actions of the liposomal enzymes. The oxidation rate of the CLG reaction increased to 1.1 mM-glucose/(hmM-lipid) at the acidic pH in the internal phase of liposome and the neutral pH in the external one corresponding to the optimal pH conditions for the activities of glucose oxidase and catalase, respectively. The oxidation rate catalyzed by the CLG could be controlled by adding sublytic concentrations of cholate to increase permeability of the liposome membrane to glucose. The catalase-conjugated liposomal system is potentially utilized for controlling the rate of reactions catalyzed by a variety of oxidases.