Spatial Distribution of Malate Dehydrogenase in Chitosan Scaffolds

Abstract

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.

Controlled Deposition of Structured Polymer Films: Chemical and Rheological Factors in Chitosan Film Formation

“…[1][2][3] Because of chitosan's demonstrated ability to immobilize enzymes in scaffold electrodes 1,2,4 and to provide enzyme stabilizing chemical microenvironments, 1,2,[4][5][6][7][8][9] new information regarding the utility of the spread-coating technique to fabricate micron thin chitosan biopolymer films, in terms of its ability to control film thickness and morphology, could prove of great benefit with regards to the field of enzyme immobilization and the production of chitosan-based biofuel cell electrodes and biosensors.…”

“…For example, deacetylated chitosan can be chemically modified to introduce amphiphilic micellar structure. [5][6][7][8][9]15,16 Introduction of an N-alkyl moiety on the water/acid soluble, hydrophilic polymer backbone yields a hydrophobic side chain, making the polymer amphiphilic, a characteristic that allows micellar structure or soluble aggregates to form. Evidence for the formation of micellar regions in chitosan have been detected via transmission electron microscopy, fluorescence spectroscopy, and static and dynamic light scattering experiments.…”

“…[5][6][7][8][9] Although the formation of micelles depends on entropy, the structure of the micellar regions of chitosan solutions depends on the relative amounts of intra-and intermolecular chemical/electronic interactions as well as key process variables, such as polymer concentration. 3,5,6,15,16 These chemical interactions include hydrogen bonding which occurs between the hydroxyl functional groups of the polymer backbone within a particular polymer strand, between neighboring strands, and with water molecules in the solution.…”

“…3,5,6,8,15,16,21,22 This is important from the perspective that for deacetylated chitosan, an enzyme is not immobilized while in solution and in the presence of the polymer (i.e., via encapsulation), but rather while the polymer condenses around the enzyme into a solid film either through freeze or air-drying (i.e., via entrapment). 7 However, upon phase separation, hydrophobically modified chitosan shows an increase in enzyme retention in the scaffold compared to deacetylated chitosan, suggesting that increased retention is related to an interaction between the enzymes and the hydrophobic groups in solution (i.e., via encapsulation). 7 These findings seem rather contradictory and unexpected, especially in light of the fact that the enzymes are assumed to prefer an aqueous environment.…”

“…7 However, upon phase separation, hydrophobically modified chitosan shows an increase in enzyme retention in the scaffold compared to deacetylated chitosan, suggesting that increased retention is related to an interaction between the enzymes and the hydrophobic groups in solution (i.e., via encapsulation). 7 These findings seem rather contradictory and unexpected, especially in light of the fact that the enzymes are assumed to prefer an aqueous environment. One possible explanation could be that the polar units are on the outside and inside of the micellar regions, with the hydrophobic groups in between them, much like a lipid bilayer, and the enzyme is on the inside surrounded by a shell of polar moieties.…”