title: Coenzyme-like ligands for affinity isolation of cholesterol oxidase.
abstract: Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China. Electronic address: marshalxy@gmail.com.
authors: Yu
authors: Y
authors: Xin
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China.
authors: Liushen
authors: L
authors: Lu
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China.
authors: Qing
authors: Q
authors: Wang
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China.
authors: Ling
authors: L
authors: Zhang
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China.
authors: Yanjun
authors: Y
authors: Tong
authors: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu 214122, PR China.
authors: Wu
authors: W
authors: Wang
mesh_terms: D002789:Cholesterol Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003067:Coenzymes / Q000378:metabolism; D008024:Ligands
chemical_list: D003067:Coenzymes; D008024:Ligands; D002789:Cholesterol Oxidase
keywords: Affinity isolation; Cholesterol oxidase; Coenzyme-like ligand