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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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 (T) 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 T values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest T 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.

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Optimized production of Aspergillus aculeatus URM4953 polygalacturonases for pectin hydrolysis in hog plum (Spondias mombin L) juice,