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Application of Monoliths as Supports for Affinity Chromatography and Fast Enzymatic Conversion

D. Josić, A. Buchacher

J. Biochem. Biophys. Methods 49 (2001) 153–174

Monoliths are useful chromatographic supports, as their structure allows improved mass transport. This results in fast separation. Once the ligand of interest has been immobilized, chromatographic separation can also be accomplished in affinity mode. Ligands with low molecular mass have been shown to be the easiest to immobilize. Nowadays, ligands with low molecular mass are often designed by combinatorial chemical techniques. In addition, many applications have been described where ligands with high molecular mass, such as Proteins A and G, antibodies, lectins and receptors are used.

The immobilization of an enzyme on the monolithic support creates a flow-through reactor. Small proteins, such as carbonic anhydrase, can be directly immobilized on the support. However, in the case of large molecules, the active center of the enzyme is no longer accessible at all or only to a limited degree. An improvement can be achieved by introducing a spacer, which allows maximum enzymatic conversion. Fast conversion of substrates with high molecular mass has been investigated with immobilized trypsin. It was shown that in case of high-molecular-mass substrates, the conversion rate depends very much on the flow-rate. Most applications described have been performed on an analytical or semi-preparative scale. However, the technical problems of up-scaling are close to being definitely solved, enabling enzymatic conversion on a preparative scale in the future.

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