Purificação e caracterização bioquímica de poligalacturonases de Aspergillus niger e sua aplicação na clarificação de sucos

Abstract

The application of enzymes in industries has been steadily growing due to their environmental efficiency and adaptability. Pectinases, a group of enzymes, play a crucial role in the degradation of plant cell wall polysaccharides, making carbon more accessible to microorganisms due to cell wall breakdown. Filamentous fungi, especially of the genus Aspergillus, lead the large-scale production of these enzymes, which are widely employed in the food industry, including in fruit juice clarification. In this context, the overall objective was to purify polygalacturonases produced by the fungus Aspergillus niger M2 and study their biochemical characteristics, and then apply them in fruit juice clarification. The purification of polygalacturonase produced by Aspergillus niger M2 was carried out through two chromatographic steps (DEAE-Fractogel and Phenyl-Sepharose), resulting in two polygalacturonases, P1 and P2. P1 was purified with a purification factor of 27.5 times, yielding 51.7%, and a specific activity of 41.2 U/mg of protein. P2, on the other hand, had a specific activity of 150.0 U/mg of protein, resulting in a purification factor of 100.0 times with a yield of 32.5%. Regarding the optimal activity conditions, P1 exhibited better activity at pH 4.0 and a temperature of 55 °C, while P2 reached its peak activity at pH 5.0 and a temperature of 50 °C. Furthermore, P1 retained over 90% of its residual activity after 4 hours at pH 4.0, while P2 maintained 100% of its residual activity after 2 hours at the three pH levels evaluated (3.0 – 5.0). Both enzymes retained over 70% of their residual activity after 4 hours at temperatures of 50 °C, 55 °C, and 60 °C. For kinetic parameters, both P1 and P2 showed a higher affinity for polygalacturonic acid (with KM values of 1.93 and 1.62 mg/ml, respectively), followed by citrus pectin and apple pectin. As for the juice clarification tests using polygalacturonases, eight fruit pulps were used, with the best results obtained for carambola pulp, 270.6% and 248.8% for P1 and P2, respectively. Next were white guava pulps, 183.9% for P1 and 183.1% for P2, and plantain pulp, reaching 142.0% and 142.5% for P1 and P2, respectively. Therefore, it can be inferred that polygalacturonases purified from Aspergillus niger M2 demonstrate significant potential for application in the beverage industry, playing a crucial role in the efficient and cost-effective production of fruit juices with greater clarity.