Structure/function relationships of different biopolymers (alginate, dextran, or beta-cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer beta-cyclodextrin-branched alginate (beta-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. Beta-CD-A (with or without trehalose), beta-cyclodextrin (beta-CD), or dextran with trehalose were the best protective agents during thermal treatment, while beta-CD and alginate showed a negative effect on invertase activity preservation. The beta-CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. Beta-CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns.
Industrial applications require enzymes highly stable and economically viable in terms of reusability. Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes. The immobilization of two commercial enzymes is reported here (cellulase and xylanase) using three chemical methods (adsorption, reticulation, and crosslinking-adsorption) and two polymeric supports (alginate-chitin and chitosan-chitin). The optimal pH for binding was 4.5 for cellulase and 5.0 for xylanase, and the optimal enzyme concentrations were 170 µg/mL and 127.5 µg/mL respectively, being the chitosan and the ideal support. In some cases, a low concentration of crosslinking agent (glutaraldehyde) improved stability of the immobilization process. Biotechnological characterization showed that the reusability of enzymes was the most striking finding, particularly of immobilized cellulase using glutaraldehyde, which after 19 cycles retained 64% activity. These results confirm the economic and biotechnical advantages of enzyme immobilization for a range of industrial applications.
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