Kinetic and thermodynamic characterization of a novel Aspergillus aculeatus URM4953 polygalacturonase. Comparison of free and calcium alginate-immobilized enzyme

Polygalacturonase (EC 3.2.1.15) (PG) from Aspergillus aculeatus URM4953 was covalently immobilized on calcium alginate beads using glutaraldehyde as a cross-linking agent. The immobilization conditions were optimized by a central composite design. Only the time influenced significantly PG immobilization, whose highest yield (95%) was obtained using 2% glutaraldehyde after 45 min. PG, characterized in terms of kinetic and thermodynamic parameters, displayed the same pH profile in free and immobilized forms, with two optimum pH values under acidic (4.0) and neutral (7.0) conditions. Although lowering the PG optimum temperature from 50 to 40 °C, the immobilization did not influence the enzyme stability in the range 30–40 °C, preserving more than 90% of its initial activity after 60 min. The kinetic parameters of immobilized PG revealed higher affinity for pectin than free PG, which nonetheless was slightly more efficient in pectin degradation. The thermodynamic parameters of immobilized PG thermoinactivation suggested a predominating mechanism of reversible unfolding, which was responsible for lower sensitivity to heating than free PG and allowed four successive cycles of immobilized enzyme utilization with only 33% of activity loss. Immobilized PG showed satisfactory performance and features as well as great potential for future industrial applications.