Produção de L-asparaginase por fermentação em estado sólido em diferentes tipos de biorreatores e seleção de microrganismos

Abstract

L-asparaginase is an enzyme of pharmaceutical interest and the food industry with several uses, highlighting the use of non-treatment of acute lymphoblastic leukemia (ALL) and the containment of acrylamide present in processed foods at high temperatures. Although commercial L-asparaginase is currently of bacterial origin, there is interest in promising new microorganisms capable of producing it by solid state fermentation (FES). In this work we investigated the production of L-asparaginase by FES evaluating different inert media, fungi and nutrient sources in two types of bioreactors (static and rotary). In the selection of the microorganisms, five yeasts (Pichia guilliermondii, Candida sake, Candida globosa, Cryptococcus victori and Leucosporidium scotti) and three filamentous fungi (Penicillium sp., LAMAI-505, Fusarium sp. and Monilia sitophila). Regarding the inert solid supports, the ceramic fiber, the polyurethane foam and the glass wool were evaluated. Regarding the source of nutrients, the use of glucose, L-asparagine, glycerol and a nutrient solution from the Tenébrio molitor were investigated. As regards the choice of the micro-organism, Penicillium sp. LAMAI-505 was selected for being efficient in the production of L-asparaginase and being classified as GRAS (generally recognized as safe) by the FDA (American Food and Drug Administration), so it is widely applied in the food industry and in use for production of medicines. As regards the inert medium, it was observed that the selected fungus presented better adaptation in the inert solid support of ceramic fiber to obtain L-asparaginase, where the use of glucose (10 g/L) and L-asparagine (1 g/L) as nutrient source reached activity of 1591.7 U/kg of solid support. Although the use of thermocycler (5 g/100 ml) together with the use of glycerol (1 g) was a complex biomass, it enabled the production of L-asparaginase (596.7 U/kg support solid). The application of glycerol and solution of Tenébrio molitor was carried out in two ways: a) glycerol applied directly to the solid inert support, under optimized conditions of pH (1,2), concentration of nutrient source (7.5 g of glycerol and 15 g/100 ml of Tenébrio molitor) and time (48 hours) reached activity of 3391.7 U/kg; b) glycerol applied directly to the nutrient solution under optimized conditions of pH (1,2), concentration of the nutrient source (15 g/L glycerol and 10 g/100 mL of Tenébrio molitor) and time (48 hours) , reached activity of 2666.7 U/kg of solid support. In this way, it was chosen to use in FES with solid inert support the glycerol applied directly to the nutrient solution, due to the great economy added materials, being used in the rotary bioreactor (maintained at 5 rpm), which in optimized condition obtained L activity -asparaginase of 2758.3 U/kg of solid support.