Osmar Soares da Silva

Thermodynamic investigation of an alkaline protease from Aspergillus tamarii URM4634: A comparative approach between crude extract and purified enzyme

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°u=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-1, while that of purified protease from 0.0099 to 0.0235min-1. 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*d=49.7 and 28.8kJ/mol), enthalpy (ΔH*d=47.0 and 26.1kJ/mol), entropy (ΔS*d=-141.3 and -203.1J/molK) and Gibbs free energy (92.6≤ΔG*d≤96.6kJ/mol and 91.8≤ΔG*d≤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.

Thermodynamic and kinetic studies on pectinase extracted from Aspergillus aculeatus: Free and immobilized enzyme entrapped in alginate beads

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-1 for the free enzyme and from 0.0017 to 0.0700 min-1 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⁎d = 85.1 and 101.6 kJ·mol-1), enthalpy (82.59 ≤ ΔH⁎d ≤ 82.34 kJ·mol-1 and 99.11 ≤ ΔH⁎d ≤ 98.86 kJ·mol-1), entropy (-63.26 ≤ ΔS⁎d ≤ -63.85 J·mol-1·K-1 and -5.50 ≤ ΔS⁎d ≤ -5.23 J·mol-1·K-1) and Gibbs free energy (101.8 ≤ ΔG⁎d ≤ 104.7 kJ·mol-1 and 100.6 ≤ ΔG⁎d ≤ 102.0 kJ·mol-1). 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.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634

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 Vmax = 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.

Biophysical, photochemical and biochemical characterization of a protease from Aspergillus tamarii URM4634

Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N-formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (Tm) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed Tm values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest Tm was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. The acquired data permits to select the best physicochemical parameters to secure the optimal activity and stability when used in biotechnological applications. Furthermore, the conformal changes induced by temperature in the protein are directly correlated with its level of biochemical activity.