期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 52, 期 1, 页码 13-19出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2012.09.001
关键词
D-Amino acid oxidase; Oligolamellar phospholipid vesicles; Unilamellar vesicles; Enzyme activity; Enzyme thermal stability
资金
- Japan Society for the Promotion of Science (JSPS) [23760751, 21246121]
- Grants-in-Aid for Scientific Research [23760751] Funding Source: KAKEN
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 degrees C compared to that of free DAO at the fixed enzyme concentration of 3.29 mu g/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. (C) 2012 Elsevier Inc. All rights reserved.
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