Specific and reversible immobilization of NADH oxidase on functionalized carbon nanotubes.
Specific and reversible immobilization of NADH oxidase on functionalized carbon nanotubes.
复制标题
DOI：
10.1016/j.jbiotec.2010.07.005
复制
发表时间：
2010-10
影响因子：
4.1
通讯作者：
Liang Wang;Li Wei;Yuan Chen;Rongrong Jiang
中科院分区：
文献类型：
--
作者：
Liang Wang;Li Wei;Yuan Chen;Rongrong Jiang
受纳米技术启发的生物催化剂系统最近在酶固定化领域引起了广泛关注。理论上，纳米材料是理想的支撑材料，因为它们可以提供酶效率决定因素的上限，例如表面积/体积比、酶负载能力和传质阻力。然而，由于酶浸出、3D结构损失和强扩散阻力，常见的固定化方法限制了这些生物催化剂的应用。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 氧化酶和功能化单壁碳纳米管之间的特异性相互作用的有效固定方法，无需酶纯化即可固定。我们从蜡样芽胞杆菌基因组中克隆了带注释的 NADH 氧化酶基因，并用编码 N 端 6× His 标签的 pET30 载体过表达。然后将带有 His 标签的 NADH 氧化酶固定到用 Nα,Nα-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，表现出良好的负载能力和稳定性，同时保持天然酶92%的最大活性。我们进一步证明固定是可逆的并且可以保留大约。几个加载周期的活动率为 92%。
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 Nα,Nα-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.