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In this work, confocal laser scanning microscopy was used to study the spatial distribution of malate dehydrogenase immobilized within three-dimensional macroporous chitosan scaffolds. The scaffolds were fabricated from solutions of native and hydrophobically modified chitosan polymer through the process of thermally induced phase separation. The hydrophobically modified chitosan is proposed to possess amphiphilic micelles into which the enzyme can be encapsulated and retained. To test this theory, we applied the immobilization procedure of Klotzbach and co-workers [J. Membr. Sci. 2006, 282 (1-2), 276-283] to solutions of fluorophore-tagged malate dehydrogenase in the presence of native and hydrophobically modified chitosan polymer and then tracked the distribution of enzymes in the resulting scaffolds using fluorescent microscopy. Results suggest that the modified chitosan does encapsulate the enzyme with a significant degree of retention and with altered distribution patterns, suggesting that hydrophobic modification of the chitosan polymer backbone does create amphiphilic regions that are capable of physically encapsulating and retaining enzymes. Commentary is also given on how this information can be correlated to enzyme activity and spatial distribution during immobilization processes.

Förster resonance energy transfer (FRET) has been used to show that the average distance between proteins decreases when the protein is immobilized within polymer scaffolds, indicating that the immobilization process is inducing aggregation.