Produção de L-asparaginase por fungos filamentosos isolados do bioma caatinga

Abstract

L-asparaginase (ASNase) is used in the treatment of acute lymphoid leukemia, the medicines contain ASNase of prokaryotic origin and the disadvantage of its use are the hypersensitivity reactions, this study had the objective of the evaluation of filamentous fungi for production of enzyme, representing a eukaryotic source. 32 filamentous fungi isolated from the Caatinga biome, through qualitative screening, using bromothymol as indicator and quantitative screening in two stages in modified Czapek Dox medium, using both culture fluid and mycelium to determine the enzymatic activity through L-aspartyl-β-hydroxamic acid (ABA) method. Characterization of Aspergillus terreus S-18 growth was carried out, the kinetic parameters {maximum growth rate (μMax), doubling time (Td) and substrate conversion factor in cells (g.g)} and optimization of biomass and enzyme production By the best producer through statistical planning divided into three blocks with repetition of the central point in two stages of cultivation, recovery of the intracellular enzyme through mechanical disruption of the mycelium, besides cultivation in an air- lift bioreactor (adapted) to evaluate the influence of the inoculum in the production of ASNase, using inoculum with spores and pre-inoculum cultured in rotativo shaker. A total of 15 fungi produced ASNase in qualitative screening, all of which produced the extracellular enzyme but the intracellular production of ASNase was higher when compared to extracellular production, and 9 fungi produced the intracellular enzyme in the quantitative screening with emphasis on intracellular production of Aspergillus Terreus S-18, which was 1.58 U.g-1 (18.2 U total) for ASNase and 0.15 U.g-1 for L- glutaminase (GLUase), showing ASNase/GLUase ratio of 10.5. In the kinetic parameters A. terreus S-18 had μMax of 0.019 h-1 , Td of 36.5 h and substrate conversion factor in cells in the growth phase of 0.9 g.g-1. In the optimization of biomass and ASNase production by A. terreus S-18, the optimum values defined for biomass production: pH 5.7 and temperature of 29 °C, already for enzyme production: pH 7.0, inoculum of 3.24E+07 spores.mL-1 and temperature of 29 °C, resulting in the production of 69.3 U total of ASNase. In the recovery of the enzyme, 0.8 U.mL-1 activity was obtained using cell disruption by glass beads and vortex. In the bioreactor cultivation, the inoculum using spores did not result in the production of extracellular and / or intracellular ASNase, and the culture using pellets of the fungus A. terreus S-18 was able to produce the intracellular enzyme, corresponding to 109.7 total U ASNase.