On-line synthesis utilizing immobilized enzyme reactors based upon immobilized dopamine beta-hydroxylase

Immobilized enzyme reactors (IMERs) based upon dopamine beta-hydroxylase (DBH) have been developed. Immobilized artificial membrane (IAM) and glutaraldehyde-P (Glut-P) stationary phases have been used to immobilize DBH. When DBH is immobilized on the Glut-P interphase the enzyme is outside the stationary phase whereas with the IAM interphase the enzyme is embedded within the interphase surroundings. The activity of each IMER and their ability for on-line hydroxylation has been investigated. The resulting IMERs are enzymatically active and reproducible. The IMERs can be utilized through the use of coupled chromatography to characterize the cytosolic (DBH-Glut-P-IMER) and membrane-bound (DBH-IAM-IMER) forms of the enzyme. The substrate is injected onto the individual IMERs and the reactants and products are eluted onto a phenylboronic acid column for on-line extraction. The substrates and products are then transported via a switching valve to coupled analytical columns. The results demonstrate that enzyme–substrate and enzyme–inhibitor interactions can be investigated with the on-line system. These IMERs can be utilized for the discovery and characterization of new drug candidates specific for the soluble form and membrane-bound form of DBH. The effects of flow-rate, contact time, pH and temperature have also been investigated.