School of Chemical & Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
J Biotechnol. 2010 Oct 1;150(1):57-63. doi: 10.1016/j.jbiotec.2010.07.005. Epub 2010 Jul 12.
Nanotechnology-inspired biocatalyst systems have attracted a lot of attention in enzyme immobilization recently. Theoretically, nanomaterials are ideal supporting materials because they can provide the upper limits on enzyme-efficiency-determining factors such as surface area/volume ratio, enzyme loading capacity and mass transfer resistance. However, common immobilization methods have limited the applicability of these biocatalysts owing to enzyme leaching, 3D structure loss, and strong diffusion resistance. Expensive enzyme purification step is also required for these methods before immobilization. In this work, we show an efficient immobilization method based on specific interaction between His-tagged NADH oxidase and functionalized single-walled carbon nanotubes without requiring enzyme purification for immobilization. We cloned the annotated NADH oxidase gene from Bacillus cereus genome and overexpressed with pET30 vector encoding N-terminal 6× His-tag. The His-tagged NADH oxidase was then immobilized onto single-walled carbon nanotubes functionalized with N(α),N(α)-bis(carboxymethyl)-L-lysine hydrate. The resulting nanoscale biocatalyst has overcome the foresaid limitations, and demonstrates good loading capacity and stability while maintaining 92% maximum activity of the native enzyme. We further demonstrate that the immobilization is reversible and can retain ca. 92% activity for a couple of loading cycles.
基于纳米技术的生物催化剂系统在酶固定化方面引起了广泛关注。从理论上讲，纳米材料是理想的支撑材料，因为它们可以提供表面积/体积比、酶载量和传质阻力等决定酶效率的因素的上限。然而，由于酶浸出、3D 结构损失和较强的扩散阻力，常见的固定化方法限制了这些生物催化剂的适用性。这些方法在固定化之前还需要昂贵的酶纯化步骤。在这项工作中，我们展示了一种基于 His 标记的 NADH 氧化酶与功能化单壁碳纳米管之间的特异性相互作用的有效固定化方法，无需进行酶纯化即可进行固定化。我们从蜡状芽孢杆菌基因组中克隆了注释的 NADH 氧化酶基因，并通过编码 N 端 6×His 标签的 pET30 载体过表达。然后，将 His 标记的 NADH 氧化酶固定在 N(α)、N(α)-双(羧甲基)-L-赖氨酸水合物功能化的单壁碳纳米管上。所得纳米级生物催化剂克服了上述限制，在保持天然酶 92%最大活性的同时，表现出良好的负载能力和稳定性。我们进一步证明了固定化是可逆的，并可以在几次负载循环中保留约 92%的活性。
