It is here reported the preparation and comparison of different monolithic micro-IMERs (Immobilized Enzyme Reactors) containing covalently immobilized human recombinant acetylcholinesterase (AChE). In particular, following previous studies on AChE-IMERs (1,2), object of this work was the covalent immobilisation of the target enzyme on disk-shaped columns of smaller dimensions (6 x 2 mm, Bed Volume = 56 mL) to increase the enzyme density on the matrix surface. Since the surface chemistry of the chosen chromatographic support will influence the yield of immobilisation as well as the environment in which the target enzyme will be inserted, the choice of an appropriate support for IMERs development represents an important issue. Moreover, “aspecific” interactions between matrix and substrate and inhibitors will also depend on the chosen material. In this context, CIM disks with different chemistries were chosen for AChE immobilisation, in particular, ethylendiamino (EDA), epoxy and CDI-CIM mini disks were selected. Moreover the introduction of an appropriate spacer arm was evaluated when required. On-line automated HPLC inhibition studies on a selected series of AChE inhibitors were performed; Aspecific interactions were evaluated and results obtained for the same compound on different CIM-based IMERs were compared. Results showed that purposely pre-derivatized CDI-based IMER gave promising eluting profile and low aspecific interactions and may therefore represent a valid chromatographic support for the development of monolithic micro-IMERs. (1) M. Bartolini, V. Cavrini, V. Andrisano: Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies. J Chromatogr A 2004, 1031: 27-34. (2) M. Bartolini, V. Cavrini, V. Andrisano: Choosing the right chromatographic support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery. J Chromatogr A. 2005, 1065:135-44.
MICRO-IMMOBILIZED ENZYME REACTORS: EVALUATION OF THE APPROPIATE CHROMATOGRAPHIC SUPPORT
BARTOLINI, MANUELA;PIAZZI, LORNA;ANDRISANO, VINCENZA
2008
Abstract
It is here reported the preparation and comparison of different monolithic micro-IMERs (Immobilized Enzyme Reactors) containing covalently immobilized human recombinant acetylcholinesterase (AChE). In particular, following previous studies on AChE-IMERs (1,2), object of this work was the covalent immobilisation of the target enzyme on disk-shaped columns of smaller dimensions (6 x 2 mm, Bed Volume = 56 mL) to increase the enzyme density on the matrix surface. Since the surface chemistry of the chosen chromatographic support will influence the yield of immobilisation as well as the environment in which the target enzyme will be inserted, the choice of an appropriate support for IMERs development represents an important issue. Moreover, “aspecific” interactions between matrix and substrate and inhibitors will also depend on the chosen material. In this context, CIM disks with different chemistries were chosen for AChE immobilisation, in particular, ethylendiamino (EDA), epoxy and CDI-CIM mini disks were selected. Moreover the introduction of an appropriate spacer arm was evaluated when required. On-line automated HPLC inhibition studies on a selected series of AChE inhibitors were performed; Aspecific interactions were evaluated and results obtained for the same compound on different CIM-based IMERs were compared. Results showed that purposely pre-derivatized CDI-based IMER gave promising eluting profile and low aspecific interactions and may therefore represent a valid chromatographic support for the development of monolithic micro-IMERs. (1) M. Bartolini, V. Cavrini, V. Andrisano: Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies. J Chromatogr A 2004, 1031: 27-34. (2) M. Bartolini, V. Cavrini, V. Andrisano: Choosing the right chromatographic support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery. J Chromatogr A. 2005, 1065:135-44.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
