Produção, purificação e caracterização bioquímica de pectinases de Aspergillus japonicus e Thermoascus aurantiacus: aplicação da enzima de A. japonicus na clarificação de sucos de frutas

Abstract

The development of innovative and sustainable alternatives to add value to agricultural and food waste (lignocellulosic biomass) is needed worldwide. Lignocellulosic biomass is a source of renewable resources because it is an organic matter made up of a complex matrix of polysaccharides, including cellulose, hemicellulose and pectin. Pectinases are a complex group of enzymes that degrade pectic substances widely used by industries, mainly in the food industry. In this context, the general objective of this study was the production of pectinases by two fungi (Aspergillus japonicus and Thermoascus aurantiacus) using low-cost substrates (agribusiness residues/products), as well as the purification and biochemical characterization of the enzymes, followed by immobilization and application of pectinase from A. japonicus in the clarification of fruit juices. In this context, the general objective was to study the production of pectinases by the fungi Aspergillus japonicus and Thermoascus aurantiacus using low-cost substrates and to analyze their applications in the clarification of fruit juices. The best pectinase production by T. aurantiacus was in SSC with cassava flour from Rondonópolis/MT with 26.7 U/g of dry substrate (or 2.67 U/mL). And for A. japonicus it was observed that the highest production of pectinase was with passion fruit peel in SSC (30 U/g of dry substrate or 3 U/mL). For T. aurantiacus, 96 hours of growth was the best time, while for A. japonicus it was 48 hours. In evaluating the effects of pH and temperature on enzyme activity, T. aurantiacus had pH 4.0 and 70 °C as optimal parameters, and A. japonicus pH 4.0 and 60 °C. In the stability evaluation, pectinase from A. japonicus (crude extract) was completely stable for 4 hours at all tested pHs. About thermostability, pectinase from A. japonicus remained stable for 6 hours at 25 °C, and after 24 hours of testing it still maintained 74% of the initial activity. At 50 °C the enzyme maintained 71% activity for 6 hours. The pectinase (crude extract) of A. japonicus was evaluated in the clarification of 13 pulps and was superior to the commercial pectinase (Pectinex) in all evaluated fruits. The best clarification obtained using 3 U/mL of crude pectinase from A. japonicus was in mango (Haden) with 85.56%, while Pectinex clarified only 50.89%. With apple (Argentina), pectinase from A. japonicus was 5 times more efficient in clarification than Pectinex, with 66.32% and 12.67% clarification, respectively. T. aurantiacus pectinase was purified in two chromatographic steps (DEAE-fractogel and Sephacryl S-200), with specific activity of 75.7 U/mg protein, resulting in a 10-fold purification with 21% enzyme recovery. And pectinase from A. japonicus was also semi-purified in two chromatographic steps (DEAE-fractogel and fenil-sepharose), resulting in a 2.9-fold purification with 81% enzyme recovery and a specific activity of 7.9 U/mg protein, exhibiting a molecular weight of about 40 kDa (named as PGAj). In mass spectrometry (LC-MS/MS) of PGAj a polygalacturonase of 29.99 kDa was the most abundant. The optimum pH and temperature for PGAj activity were pH 4.0 and 55 °C, respectively. Furthermore, the PGAj enzyme retained over 90% of its initial activity for 4 hours at pH 4.0, 5.0 and 6.0. The enzyme maintained 83% of residual activity after 20 min at 50 °C. In the specificity test, PGAj had citrus pectin as the preferred substrate, followed by apple pectin and polygalacturonic acid. For the juice clarification tests by PGAj, 13 pulps were also used, where the best result was obtained with mango pulps (Palmer and Tommy), with 65% and 41%, while Pectinex clarified only 49% and 21%, respectively. Then the white guava, the banana “nanica” and the gala apple, had 40%, 11% and 9.4% of clarification, respectively. The best result of immobilization obtained was with 2% sodium alginate, using 0.1 M CaCl2. In reuse tests, immobilized PGAj maintained 100% activity after 6 reaction cycles using 1% pectin as substrate. It is concluded that crude and semi-purified pectinase from A. japonicus showed potential for application in beverage industries, to contribute to an efficient and economical production of clearer fruit juices.