Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay.
An online immobilized glucose oxidase (GOx) capillary microreactor was developed based on an enzymatic redox reaction with 1,4-benzoquinone as an acceptor of electrons, replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation. A high efficiency of enzymolysis was obtained at 1 mg ml⁻¹ 1,4-benzoquinone for 5 min of incubation at 25°C, and baseline separation of the substrate and product could be achieved with a resolution of 3.85 by employing 20mM phosphate buffer (pH 8.0) containing 40 mg ml⁻¹ sulfated β-cyclodextrin as an additive, a constant voltage of 15 kV, and a detection wavelength of 220 nm. In addition, an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag⁺ and Cu²⁺ used as model inhibitors. The results indicate that Ag⁺ (IC₅₀=69.16 μM) has a markedly higher inhibitory effect than Cu²⁺ (IC₅₀=1.33 mM). The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors.
College of Science, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.
Siming
S
Wang
Ping
P
Su
Yi
Y
Yang
D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology; D016227:Benzoquinones / Q000737:chemistry*; D019149:Bioreactors; D003300:Copper / Q000737:chemistry; D019075:Electrophoresis, Capillary; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D005947:Glucose / Q000032:analysis*; D005949:Glucose Oxidase / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D015203:Reproducibility of Results; D012834:Silver / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000737:chemistry
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D047392:beta-Cyclodextrins; D012834:Silver; C004532:quinone; D003300:Copper; D005949:Glucose Oxidase; D005947:Glucose; D010100:Oxygen