Yoshimoto Makoto, Wang Shaoqing, Fukunaga Kimitoshi, Fournier Didier, Walde Peter, Kuboi Ryoichi, Nakao Katsumi
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube, 755-8611, Japan.
Biotechnol Bioeng. 2005 Apr 20;90(2):231-8. doi: 10.1002/bit.20422.
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.
在我们之前的工作中（Wang等人，[2003]《生物技术与生物工程》83:444 - 453）制备的固定化含葡萄糖氧化酶脂质体（IGOL），通过将来自大肠杆菌的通道蛋白OmpF整合到脂质体膜中，以及不仅将黑曲霉的葡萄糖氧化酶（GO），还将过氧化氢酶（CA）包裹在脂质体的水相内部，其反应活性在此得到了显著提高。CA用于分解脂质体内部葡萄糖氧化反应中产生的过氧化氢。OmpF的存在增强了葡萄糖分子从脂质体外部到内部的运输。在工作的第一步，制备并表征了含有GO和CA的脂质体（GOCAL）。发现在40℃时脂质体膜对脂质体内部的CA具有显著的保护作用；对于GOCAL，在孵育72小时后剩余的CA活性超过60%，而游离CA的剩余活性不到20%。在第二步中，将OmpF整合到GOCAL膜中，导致形成嵌入OmpF的GOCAL（缩写为GOCAL - OmpF）。由于跨脂质体双层的葡萄糖渗透增加，GOCAL - OmpF内部的GO活性与GOCAL内部相比提高了17倍，且没有GO或CA从脂质体中泄漏。估计最佳系统平均每个脂质体含有五个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于40℃连续15小时空气氧化过程中跟踪剩余的GO活性以进行重复使用，来检测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）表现出更高的反应活性和可重复使用性。即使催化剂重复使用四次，IGOCAL - OmpF仍能实现约80%的葡萄糖转化率，而对于IGOL和IGOL - OmpF，相应的转化率分别约为60%和20%。由于缺乏CA，IGOL - OmpF稳定性较差，导致GO受到严重抑制。
