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This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β‐fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45–65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol−1, and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3–290.4 kJ mol−1, 568.7–571.0 J mol−1 K−1, and 97.9–108.8 kJ mol−1, respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo‐oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long‐term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.

The thermostable crude proteolytic extract and purified protease produced by Aspergillus tamarii URM4634 were investigated at different temperatures. The activity results were used to estimate the activation energy of the hydrolysis reaction catalyzed by crude extract and purified protease (E*=34.2 and 16.2kJ/mol) as well as the respective standard enthalpy variations of reversible enzyme unfolding (ΔH°=31.9 and 13.9kJ/mol). When temperature was raised from 50 to 80°C in residual activity tests, the specific rate constant of crude proteolytic extract thermoinactivation increased from 0.0072 to 0.0378min, while that of purified protease from 0.0099 to 0.0235min. These values, corresponding to half-life decreases from 96.3 to 18.3min and from 70.0 to 29.5min, respectively, enabled us to estimate the activation energy (E*=49.7 and 28.8kJ/mol), enthalpy (ΔH*=47.0 and 26.1kJ/mol), entropy (ΔS*=-141.3 and -203.1J/molK) and Gibbs free energy (92.6≤ΔG*≤96.6kJ/mol and 91.8≤ΔG*≤98.0kJ/mol) of thermoinactivation. Such values suggest that this protease, which proved to be highly thermostable in both forms, could be profitably exploited in industrial applications. To the best of our knowledge, this is the first comparative study on thermodynamic parameters of a serine protease produced by Aspergillus tamarii URM4634.

The kinetics and thermodynamics of Aspergillus aculeatus pectinase, either free or immobilized in alginate beads, were investigated. Pectinase immobilization ensured an enzyme immobilization yield of 59.71%. The irreversible denaturation of pectinase in both preparations was evaluated at temperatures ranging from 30 to 60 °C. When temperature was raised, the first-order thermal denaturation constant increased from 0.0011 to 0.0231 min for the free enzyme and from 0.0017 to 0.0700 min for the immobilized one, respectively. The results of residual activity tests enabled us to estimate, for denaturation of both free and immobilized pectinase, the activation energy (E = 85.1 and 101.6 kJ·mol), enthalpy (82.59 ≤ ΔH ≤ 82.34 kJ·mol and 99.11 ≤ ΔH ≤ 98.86 kJ·mol), entropy (-63.26 ≤ ΔS ≤ -63.85 J·mol·K and -5.50 ≤ ΔS ≤ -5.23 J·mol·K) and Gibbs free energy (101.8 ≤ ΔG ≤ 104.7 kJ·mol and 100.6 ≤ ΔG ≤ 102.0 kJ·mol). The integral activity of a continuous system using the free and immobilized enzyme was also predicted, whose results indicated a satisfactory enzyme long-term thermostability in both preparations at temperatures commonly used to clarify juice. These results suggest that both free and immobilized pectinase from A. aculeatus may be profitably exploited in future food industrial applications, with special concern to the immobilized enzyme because of its reusability.

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