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258 Benčina et al.
1. Introduction
Enzymes immobilized on solid-phase matrices have found various applications
in biotechnology, molecular biology and molecular diagnostics and can
serve as industrial catalysts and as specific reagents for analytical procedures. The
advantages of using immobilized enzymes instead of an enzyme solution include
increased stability and an opportunity to work with a continuous system over long
periods of time. A wide range of supports have been utilized for immobilization
among which particle-based supports are the most commonly implemented. The
typeofsupportusedforimmobilizationisoneofthekeyconsiderationsinpractical
application due to different immobilization efficiency, ligand utilization and mass
transfer regime. The mass transfer between the mobile phase and the stationary
phase has a pronounced effect on the performance. In the case of particulate porous
supports, the substrate has to diffuse from the mobile phase into the pores in order
to reach the catalytic sites of the immobilized enzyme. Because the diffusion,
especially for large molecules, is commonly slower than the reaction process at
the active site, the overall kinetic behavior of the immobilized enzyme is governed
by mass transfer, causing a decrease in efficiency.
To overcome this drawback, a new group of supports called monoliths was
introduced (1). Contrary to conventional stationary phases that are in the form
of a few micrometer particles, monoliths are made of a single piece of porous
material. Pores are highly interconnected forming a channel network through
which the mobile phase flows. As the main transport mechanism is convection,
mass transfer resistance can be neglected under operating conditions. Consequently,
the overall behavior of the immobilized ligand reflects its intrinsic
reaction kinetics. Therefore, such an immobilized system is expected to allow
higher throughput because of higher enzyme efficiency, especially pronounced
for macromolecular substrates having low mobility.
Among different types of monoliths, methacrylate-based monoliths were
most frequently used for immobilization of various ligands. As such, they
were used either as an affinity support for purification of target compounds
(2,3) or as bioreactors (2,4,5). In this work, some examples of macromolecular
bioreactors based on Convective Interaction Media (CIM) ® (CIM is a registered
trademark of BIA Separations, Ljubljana, Slovenia) supports (methacrylatebased
monoliths) are presented.
2. Materials
1. CIM Convective Interaction Media ® epoxy groups containing poly (glycidyl
methacrylate-co-ethylene dimethacrylate) monolithic columns (BIA Separations)
with a diameter of 12 mm and thickness of 3 mm (monolith volume 0.34 ml)
having median pore size of approximately 1.5 or 6 μm (CIM epoxy disk).