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.
As a result of the interest that exists in the liberation of aromas in young wines, we obtained some different enzymatic extracts (purified extract, P; lyophilized purified extract, LP; immobilized purified extract, IP; and immobilized lyophilized purified extract, ILP) with beta-glucosidase activity from Debaryomyces pseudopolymorphus, which excreted the enzyme in the growth medium. The extracts were added to natural glycosides isolated from different grape varieties. The results were compared with the effect of seven commercial enzyme preparations (CEP), obtained from molds used in wine making. It was shown that some yeast extracts had effects similar to those of the CEP, and the next step was to use them on wine samples elaborated in the laboratory. The effect was studied at 9 and 16 days of contact, quantifying both the precursors that were retained and the liberated terpenes. The results were compared with a control wine (without any extract) and with wine treated with a commercial enzyme preparation specially indicated for the liberation of aromas. It was observed that the enzymatic extracts from Db. pseudopolymorphus hydrolyzed the precursors in wine and that they could compete with the commercial preparations since the liberation was produced in even less time.
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