title: A rapid in situ immobilization of D-amino acid oxidase based on immobilized metal affinity chromatography.
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-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.
authors: Department of Chemical Engineering, Tsinghua University, Beijing, China.
authors: Jixian
authors: J
authors: Hou
authors: Quan
authors: Q
authors: Jin
authors: Jing
authors: J
authors: Du
authors: Qiang
authors: Q
authors: Li
authors: Qipeng
authors: Q
authors: Yuan
authors: Jichu
authors: J
authors: Yang
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004800:Enzymes, Immobilized / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D003605:D-Amino-Acid Oxidase