Hou Jixian, Jin Quan, Du Jing, Li Qiang, Yuan Qipeng, Yang Jichu
Department of Chemical Engineering, Tsinghua University, Beijing, China.
Bioprocess Biosyst Eng. 2014 May;37(5):857-64. doi: 10.1007/s00449-013-1056-6. Epub 2013 Dec 11.
A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of D-amino acid oxidase (DAAO) with binding-enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni(2+) support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.
基于固定化金属亲和色谱（IMAC）的传统分离技术，开发了一种快速原位固定化方法，并以具有增强结合能力的Heli-tag（His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly）的D-氨基酸氧化酶（DAAO）为例进行了研究。成功构建了重组大肠杆菌菌株JM105（Δase）/pGEMK-R-DAAO-Heli，以合成嵌合蛋白DAAO-Heli。无需额外的纯化步骤，带有标签的酶DAAO-Heli可以直接固定在EP-IDA-Ni(2+)载体上，纯度为90%，DAAO活性超过70 U/g载体。实验结果表明，固定化的DAAO-Heli的热稳定性比游离酶高73倍。此外，经过100次分批催化循环后，它仍保留了67%的初始活性，其操作稳定性比之前IMAC固定化的DAAO-His提高了36倍。此外，环氧（EP）载体可以通过简单的步骤轻松回收并重复使用，这可以显著降低固定化成本。
