Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase.
Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase.
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DOI：
10.1002/bit.20422
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发表时间：
2005-04
影响因子：
3.8
通讯作者：
M. Yoshimoto;Shaoqing Wang;K. Fukunaga;D. Fournier;P. Walde;R. Kuboi;K. Nakao
中科院分区：
文献类型：
--
作者：
M. Yoshimoto;Shaoqing Wang;K. Fukunaga;D. Fournier;P. Walde;R. Kuboi;K. Nakao
我们之前的工作（Wang et al. [2003] Biotechnol Bioeng 83:444-453）中制备的固定化葡萄糖氧化酶脂质体（IGOL）的反应性通过将来自大肠杆菌的通道蛋白OmpF掺入脂质体膜以及通过将葡萄糖氧化酶（GO）和过氧化氢酶包埋在脂质体的水性内部而得到显着改善。 (CA)，均来自黑曲霉。 CA用于分解脂质体内葡萄糖氧化反应产生的过氧化氢。 OmpF 的存在增强了葡萄糖分子从脂质体外部到内部的转运。在工作的第一步中，制备并表征了含有 GO 和 CA (GOCAL) 的脂质体。发现40℃时脂质体膜对脂质体内的CA有显着的保护作用；孵育72小时后，GOCAL的剩余CA活性超过60%，而游离CA则低于20%。第二步，将 OmpF 掺入 GOCAL 膜中，形成 OmpF 嵌入的 GOCAL（缩写为 GOCAL-OmpF）。由于脂质体双层的葡萄糖渗透增加，GOCAL-OmpF 内的 GO 活性与 GOCAL 内相比增加了 17 倍，且没有任何 GO 或 CA 从脂质体中渗漏。据估计，最佳系统每个脂质体平均含有 5 个 OmpF 分子。最后，将 GOCAL-OmpF 共价固定到壳聚糖凝胶珠中。通过跟踪葡萄糖转化率的变化以及在气升式生物反应器中在 40 摄氏度下重复使用连续 15 小时空气氧化中的剩余 GO 活性来检查这种新型生物催化剂 (IGOCAL-OmpF) 的性能。 IGOCAL-OmpF 比 IGOL 以及含有 OmpF 的 IGOL (IGOL-OmpF) 显示出更高的反应性和可重复使用性。即使催化剂重复使用四次，IGOCAL-OmpF 也能产生约 80% 的葡萄糖转化率，而 IGOL 和 IGOL-OmpF 的相应转化率分别约为 60% 和 20%。由于缺乏 CA，IGOL-OmpF 不太稳定，导致 GO 受到严重抑制。
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.