Fabrication of enzyme reactor utilizing magnetic porous polymer membrane for screening D-Amino acid oxidase inhibitors.
In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Kmwas 1.10mM, Vmaxwas 23.8mMmin-1) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.
Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China.
Jun Fang
JF
Jiang
Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China. Electronic address: qiaojuan@iccas.ac.cn.
Juan
J
Qiao
Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China.
Xiao Yu
XY
Mu
Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
Myeong Hee
MH
Moon
Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China. Electronic address: qili@iccas.ac.cn.
Li
L
Qi
D019149:Bioreactors; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008280:Magnetics; D008566:Membranes; D008715:Methionine / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D016062:Porosity
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008024:Ligands; D011108:Polymers; D008715:Methionine; C417983:DAO protein, human; D003605:D-Amino-Acid Oxidase
Covalently immobilized enzyme reactor; D-amino acid oxidase inhibitors screening; Magnetic porous polymer membrane