Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

Abstract

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.

The antioxidant effect of boric acid and CoQ10 on pulmonary fibrosis in bleomycin induced rats

“…Catalase (CAT) is essential to protect aerobic organisms from the toxic effects of H2O2. Catalase was also utilised in an enzymatic oxidation reaction to depress the deactivation of the relevant enzyme and/or the side reaction due to H2O2 that was produced (Yoshimoto et al 2005). Özyurt et al (2004) reported the significant decrease in CAT and SOD activities in lung tissue in bleomycine-induced lung fibrosis in rats.…”

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

“…In an elegant approach, the catalase-conjugated liposome encapsulating glucose oxidase was used to prevent oxidase inactivation by hydrogen peroxide [151,152]. The reactivity of immobilized glucose oxidase-containing liposomes was considerably improved by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane [153].…”

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

“…The other system that has received attention is the couple bacterhodopsin/ATP synthase (both membrane proteins) that have been reconstituted in liposomes at the aim of producing ATP after irradiation (Freisleben et al, 1995;Pitard et al, 1996;Choi and Montemagno, 2007). With respect to the control of vesicle membrane permeability, it is worth mentioning the increase of passive permeability of detergent-doped membranes (the concentration of detergent is below the lytic regime, Treyer et al, 2002), and the use of -hemolysis (Noireaux & Libchaber, 2004) or porines (Graff et al, 2001;Vamvakaki et al, 2005;Yoshimoto et al, 2005) as channel-forming agents. The next step along the road map for SSMC construction is the realization of nucleic acidbased reactions inside liposomes.…”

“…The membrane acts as a boundary to confine the interacting internalized molecules, so that a "unit" is defined. Moreover, its semi-permeable character (possibly modulated thanks to the insertion of membrane proteins acting as selective pores, as in the case of hemolysin (Noireaux & Libchaber, 2004) or porins (Graff et al, 2001;Vamvakaki et al, 2005;Yoshimoto et al, 2005) allows the material exchange between the SSMC and its environment (nutrients uptake, waste release).…”