College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China.
College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China; State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing 210009, China; Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China; School of Chemical Engineering, The University of Queensland, St Lucia, Queensland 4072, Australia.
Colloids Surf B Biointerfaces. 2018 Apr 1;164:155-164. doi: 10.1016/j.colsurfb.2018.01.033. Epub 2018 Jan 31.
The development of an orientation immobilization technique via affinity between polyhistidine tags and metal ions aims at maintaining biocatalytic activity of the enzymes. In this work, to tackle the issue of the immobilization of adenylate cyclase (AC), a simple and effective approach of synthesizing iminodiacetic acid (IDA)-Ni particles was applied for simultaneously purifying and immobilizing his-tagged AC. We chose agarose particles as carriers, and then decorated them with IDA, leading to the formation of a coordination combination of Ni. The porous carriers with a large pore size of 50 nm and a specific surface area of 45.8 m/g exhibited favorable enzymatic activity and loading capacity. The optimal pH of the immobilized enzyme increased from 8.0 to 9.0 and the optimal temperature increased from 30 °C to 35 °C, compared to the free AC. Moreover, the immobilized AC retained a residual activity of approximately 80% after storing it at 25 °C for 48 h, whereas only 40% of the activity was left in the free AC at the same conditions. Maximum yield of cyclic adenosine-3', 5'- monophosphate (cAMP) reached up to the summit of the reaction. The immobilized AC by affinity adsorption will provide a promising route for the industrial production of cAMP.
本研究旨在通过组氨酸标签与金属离子之间的亲和作用开发一种定向固定化技术，以保持酶的生物催化活性。在这项工作中，为了解决腺苷酸环化酶（AC）的固定化问题，我们采用了一种简单有效的亚氨二乙酸（IDA）-Ni 粒子合成方法，用于同时纯化和固定 his 标记的 AC。我们选择琼脂糖颗粒作为载体，然后用 IDA 对其进行修饰，形成 Ni 的配位组合。多孔载体的孔径为 50nm，比表面积为 45.8m/g，具有良好的酶活性和负载能力。与游离 AC 相比，固定化酶的最适 pH 从 8.0 增加到 9.0，最适温度从 30°C 增加到 35°C。此外，固定化 AC 在 25°C 下储存 48 小时后仍保留约 80%的剩余活性，而在相同条件下，游离 AC 的活性仅保留 40%。环腺苷酸-3'，5'-单磷酸（cAMP）的最大产率达到了反应的峰值。通过亲和吸附固定化的 AC 将为 cAMP 的工业生产提供一条有前途的途径。
