Produção e extração de proteases colagenolíticas obtidas de Aspergillus heteromorphus URM 0269 e sua imobilização em alginato

Abstract

This work aimed to produce, immobilize in calcium alginate, extract in two-phase aqueous systems (PEG/Citrate) and characterize collagenolytic proteases obtained from Aspergillus heteromorphus URM 0269. Therefore, in chapter I, a solid state fermentation was carried out using a factorial design 22; the best production condition was: 3g of wheat bran, 20% moisture, submitted to 30°C after 96 hours of fermentation, with a proteolytic and collagenolytic activity of 41.36 U/mL and 401.06 U/mL, respectively. The collagenolytic protease contained in the enzymatic extract was immobilized by imprisonment in alginate beads, and a factorial design 22 was carried out where the best conditions were obtained (0.6M CaCl2; 4% sodium alginate) for further analysis of yield, reuse, stability of storage and biochemical characterization. In this best test there was a yield of 82.82% for proteolytic activity and 94.58% of collagenolytic activity. This immobilized enzyme retained more than 40% of the residual activity in the third cycle and showed only 36.82% loss of activity after 7 days of storage at 4°C. Optimum pH and temperature, as well as stability, were also analyzed. In chapter II, a factorial design (24) was carried out for purification of the collagenolytic protease using the aqueous two-phase system (ATPS), where it was possible to evaluate the interaction of the independent variables: molar mass of PEG (MPEG), concentration of PEG (CPEG), sodium citrate concentration (CCIT) and pH. Subsequently, the protease extracted by ATPS was used for the hydrolysis of azocasein and characterized in terms of biochemical, kinetic and thermodynamic parameters. The enzyme was preferentially partitioned for the PEG-rich phase whose greatest purification and recovery factor (PF = 7,8 and Y = 157,5%) was obtained using MPEG 8000 g / mol, CPEG 24%, CCIT 15% and pH 8,0. Although the enzyme acted ideally at 50°C and pH 8.0, it was more stable at lower temperatures (10-30 °C) and acidic pH conditions (5.0-6.0). The purified enzyme was inhibited by PMSF, classifying it as a serine protease. The kinetic activation parameters for azocasein hydrolysis revealed greater affinity for azocasein (KM = 2.8 mg / mL) with a maximum catalysis rate of 45.0 U/mL. The reaction activation energy and the standard enthalpy variation of the enzyme development were 24.2 and 54.2 kJ/mol, respectively. Protease catalyzed hydrolysis at 25°C showed Gibbs free energy of activation, enthalpy and entropy of 65.8 kJ/mol, 21.8 kJ/mol and -146.7 J/mol.K, respectively. The thermodynamic parameters of the protease thermoactivation in the ATPS suggest a predominant reversible unfolding mechanism, since the inactivation of Gibbs free energy increased from 91.0 to 102.3 kJ/mol. Solid state fermentation was effective with high enzymatic production. The immobilization of collagenolytic protease in calcium alginate, proved to be an efficient method in the yield, storage and reuse of the enzyme. Through a fast and economical process, the enzyme was purified by ATPS, allowing the removal of contaminants in the enzymatic extract obtained from Aspergillus heteromorphus URM0269. These results indicate the potential of collagenolytic protease to be explored in biotechnological applications, such as in the tannery and detergent industries.