CASTRILLO Maria lorena
congresos y reuniones científicas
Biochemical characterization of the optimal reaction temperature and pH of the main mycolytic enzymes with biocontrol capacity of the Escovopsis primorosea HMP9 strain
Workshop; 3er Encuentro y 1er Workshop de la Red Argentina de Tecnología Enzimática; 2021
In the province of Misiones, one of the main pests that affects the primary forest sector are leaf cutter ants. Its forage activity consists of cutting and transporting fresh plant material to its nest, on which the fungus Leucoagaricus gongylophorus (Basidiomycota: Agaricales) grows, which is its main source of food. On the other hand, the fungi of the genus Escovopsis (Ascomycota: Hipocreales), are considered specialist parasites of L. gongylophorus. These mycoparasites secrete extracellular mycolytic enzymes, mainly chitinases, β-1,3-glucanases and proteases, which are capable of hydrolyzing the components of the cell walls of their host. Therefore, Escovopsis appears as a potential indirect biocontroller of leaf cutter ants. An effective way for its biotechnological application is to induce its mycolytic activity, optimizing its fermentation conditions, and biochemically characterizing the enzymes with optimized biocontrolling capacity.Therefore, the aim of this work was to determine the optimal reaction temperature and pH conditions of proteases, chitinases and β-1,3-glucanases, in optimized enzymatic supernatants of a strain of Escovopsis native to Misiones.The promising strain of Escovopsis primorosea HMP9 was used at an inoculum concentration of 4 x 106 spores/mL. Culture media previously optimized for the secretion of mycolytic enzymes were used in liquid fermentation with an initial medium pH of 4,63. As a carbon source, cell walls of L. gongylophorus (0,36 g/L) were used, and as a nitrogen source the Mandels complex whose nitrogenous components were, urea (0,1 g/L), yeast extract (1,86 g/L) and ammonium sulfate (0,21 g/L).To quantify protease activity, the azocasein method was used; and for the β-1,3-glucanases and chitinases activities, the dinitrosalicylic acid method was used. A one factor optimization design at a time was used, using as initial pHs those indicated in the standardized protocols for these enzymes. The effect of temperature on protease activity was evaluated by incubating the reaction mixtures at pH 7,4 and different temperatures; 25, 37, 45, 55, 65, 75, 85, and 95 °C; for chitinases they were incubated at pH 4,8 and temperatures of 25, 37, 45, 55 and 65 ° C; and for β-1,3-glucanases they were incubated at pH 5 and temperatures of 25, 40, 50, 60 and 70 °C. The effect of pH was determined by incubating the reaction mixtures at optimal temperature, using different buffers to vary the reaction pH; for proteases pHs 5, 6, 7, 7,4, 8 and 9 were tested; for chitinases 3, 4, 4,8, 5, 6 and 7; and for β-1,3-glucanases 3, 4, 5, 6 and 7. The other reaction conditions were kept constant and the tests were carried out in duplicate. The enzymatic activity was expressed in U/L. The data were processed using the statistical program InfoStat version 2018. An analysis of variance (ANOVA) and a test of difference between means were performed, using the Tukey test, with a confidence level of 95,0%.Regarding protease activity, the maximum activity (393,15 ± 8,35 U / L) was observed at 85 °C and pH 7,4, with a statistically significant difference (p < 0,05) compared to the others terms. For chitinase activity, no significant differences were observed in the temperature range from 37 to 55 °C, and between pHs 4 to 5, obtaining a higher activity (51,9 ± 2,54 U/L) at 45 °C and pH 4. For β-1,3-glucanase activity, the maximum activity (547,035 ± 6,69 U/L) was obtained at 60 °C and pH 5, with a significant difference (p < 0,05) with respect to the other conditions.These results made it possible to establish the optimum temperature and pH conditions in which enzymes with biocontrolling capacity act. Subsequently, the enzymatic stability over time of each enzyme against different pH and temperature conditions will be determined. The biochemical characterization of the optimized fungal supernatants represents an important factor for the efficient application of these mycolytic enzymes in the biological control of leaf cutter ants.