A rapid in situ immobilization of d-amino acid oxidase based on immobilized metal affinity chromatography |
| |
| Authors: | Jixian Hou Quan Jin Jing Du Qiang Li Qipeng Yuan Jichu Yang |
| |
| Institution: | 1. Department of Chemical Engineering, Tsinghua University, Beijing, China
2. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China
|
| |
| Abstract: | 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-Ni2+ 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. |
| |
| Keywords: | |
| 本文献已被 SpringerLink 等数据库收录！ |
|
