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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.

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.

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This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited K M = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 -11.0) during 24 h. The optimum temperature was 40˚C. The proteolytic activity was inhibited by Cu 2+ (33.98%) and Hg 2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.

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An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg and Ca, but inhibited by Zn, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and V = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

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