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.

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Pectinase extracted from Aspergillus niger was immobilized on a chitosan-coated chitin support using various methods: immobilization by adsorption (P-QQSA), adsorption on supports activated by 0.5 and 15% glutaraldehyde (w/v) (P-QQSA 0.5 and P-QQSA 15) and covalent attachment to this support using 1% glutaraldehyde (P-QQSA 1). The optimum conditions selected for immobilization were pH 4.5, incubation time of 4 h and protein concentration of 340 μg/mL. Various characteristics of the immobilized pectinase such as optimum pH, heat stability and reusability were evaluated. As a result of immobilization the enzyme's T50 increased, the best being achieved with immobilization using 15% glutaraldehyde and covalent attachment. The optimum pH of the free and immobilized enzymes were 5 (free), 4.5 (P-QQSA), 4.5-5.0 (P-QQSA 0.5) and 4-5 (P-QSA 1 and P-QQSA 15), respectively. The biocatalysts prepared retained 100% of their original catalytic activity after nine cycles of reuse. PRACTICAL APPLICATIONSPectinases hydrolyze pectin and/or pectic acid. These enzymes have widespread applications in the food industry (processing of fruits), wastewater treatment, textile industries, fruit softening and plant infection processes. The stability of these enzymes depends on the aqueous medium and is easily disrupted to the point where the enzymes cannot function appropriately. Immobilization techniques provide a promising approach to retain their stability. Various methods for immobilization of this enzyme have been described: entrapping in alginate, physical adsorption on anion resin, γ-alumina, particles and nanoparticles of silica and covalent attachment to carriers such as porous glass and nylon. However, the development of new methods and supports for immobilizing enzymes is of special importance in enzyme technology. The present article describes several methods for immobilization of pectinase in chitosan-coated chitin for use in the juice and wine industries.