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

Paper ID Volume ID Publish Year Pages File Format Full-Text
17150 42647 2013 7 PDF Available
Title
Oligolamellar vesicles for covalent immobilization and stabilization of d-amino acid oxidase
Abstract

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.

► d-Amino acid oxidase is covalently immobilized in oligolamellar vesicles. ► Oligolamellar structure of vesicles is elucidated by NBD fluorescence quenching. ► The enzyme immobilized in vesicles shows significantly high thermal stability.

Keywords
D-Amino acid oxidase; Oligolamellar phospholipid vesicles; Unilamellar vesicles; Enzyme activity; Enzyme thermal stability
First Page Preview
Oligolamellar vesicles for covalent immobilization and stabilization of d-amino acid oxidase
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issue 1, 10 January 2013, Pages 13–19
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering