Produção e Purificação de L-asparaginase por Aspergillus caespitosus CCDCA 11593

Abstract

This work aimed to study the production and purification of the enzyme L-asparaginase (EC 3.5.1.1) produced by the filamentous fungus Aspergillus caespitosus, through solid and submerged fermentation processes, using alternative substrates such as ora-pro-nobis fiber (Pereskia aculeata Miller) and whey powder, respectively. A centered face design was applied in order to optimize the enzyme production. Through the solid fermentation (FES) the influence of the parameters was evaluated: fermentation time (h), inoculum concentration (spores/ml), asparagine concentration (% m/v), temperature (° C) and substrate concentration (ora-pro-nobis fiber) on the volumetric activity of L-asparaginase (U/ml). The submerged fermentation process (FSbm) evaluated the parameters: concentration of asparagine, concentration of inoculum, temperature, fermentation time, pH and concentration of substrate (whey powder) on the enzymatic activity of L-asparaginase. The results of the statistical analyzes indicated that the best condition for the production of the enzyme by solid fermentation was obtained by using an inoculum concentration of 105 spores/ml, temperature of 25 ºC, fermentation time 120 h, 14% substrate and 1% of asparagine, obtaining 2.7498 U/mL of volumetric activity. Submerged fermentation, on the other hand, presented the best result in production conditions with 10% asparagine, 6% substrate, spore concentration 107 spores/mL, temperature of 25 ºC, pH 7 and 120 hours of fermentation, with an activity of 1, 49265 U/ml. The contour graphics were chosen to better demonstrate the interactions between the parameters that were significant in both fermentation processes. The interactions between temperature and fermentation time negatively influenced the production of L-asparaginase from FES. While in the submerged fermentation the interactions between asparagine concentration and inoculum concentration, temperature and inoculum concentration, whey concentration and temperature and pH and temperature negatively affected the enzyme production. In FSbm, the interactions between the parameters inoculum concentration and pH, inoculum concentration and substrate concentration, asparagine concentration and fermentation time, inoculum concentration and asparagine concentration, inoculum concentration and temperature, temperature and substrate concentration, temperature and pH, positively affected the volumetric activity of L-asparaginase. Then, batch adsorption tests were performed to find the best condition for the purification of the selected asparaginase obtained from the optimization of the submerged fermentation. The enzyme adsorption tests were performed using an ion exchange cryogel column functionalized with 2-aminoethanesulfonic acid, using a sodium phosphate balance buffer (0.05 mol/L) at pH 3. The purified enzyme presented an activity of 4,400 U/mg, whereas the crude extract presented 2,779 U/mg. These results showed that the production of L-asparaginase from the Aspergillus caespitosus species is promising by solid and submerged fermentation processes using alternative substrates such as ora-pro-nobis leaf fiber and whey.