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Optimal covalent immobilization of glucose oxidase‐containing liposomes for highly stable biocatalyst in bioreactor
Author(s) -
Wang Shaoqing,
Yoshimoto Makoto,
Fukunaga Kimitoshi,
Nakao Katsumi
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10684
Subject(s) - glutaraldehyde , liposome , chemistry , chromatography , immobilized enzyme , glucose oxidase , phosphatidylcholine , membrane , thermal stability , denaturation (fissile materials) , covalent bond , nuclear chemistry , biosensor , biochemistry , organic chemistry , phospholipid , enzyme
Abstract The glucose oxidase‐containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L‐α‐phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde‐activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4°C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40°C, and also by its high stability at 40°C against not only the thermal denaturation in a long‐term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small‐scale airlift bioreactor. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 444–453, 2003.