Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase.
The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.
Yoshimoto
Makoto
M
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube, 755-8611, Japan.
Wang
Shaoqing
S
Fukunaga
Kimitoshi
K
Fournier
Didier
D
Walde
Peter
P
Kuboi
Ryoichi
R
Nakao
Katsumi
K
D019149:Bioreactors; D002374:Catalase / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism*; D010084:Oxidation-Reduction; D018272:Porins / Q000378:metabolism*
D004800:Enzymes, Immobilized; C109237:OmpF protein; D018272:Porins; D005949:Glucose Oxidase; D002374:Catalase; D005947:Glucose