| Congresso Brasileiro de Microbiologia 2023 | Resumo: 852-2 | ||||
Resumo:L-asparaginase is an enzyme that catalyzes the hydrolysis reaction of the amino acid L-asparagine into aspartic acid and ammonia. The application of a new source of L-asparaginase production can contribute to reduce the hypersensitivity reactions caused by the enzyme derived from prokaryotic organisms. The aim of this work was to evaluate the interference of different cultivation conditions in the expression of the enzyme and to solubilize the inclusion bodies formed. The L-asparaginase produced by the fungus Fusarium proliferatum was cloned into an heterologous system using the expression vector pET-28a and mutated Escherichia coli as a host cell. After cell transformation, they were maintained in Luria Bertani broth containing kanamycin, under different conditions. After each cultivation time, the samples were centrifuged and the medium discarded, and these were maintained in lysis buffer containing PMSF, EDTA and Tris-HCl. Then, extraction was performed using the sonication on ice bath methodology. For the inclusion bodies solubilization assay, a 200 mL culture was prepared at 37 °C, 200 rpm, with 0.5 mM of IPTG for 12 hours. Subsequently, samples were subjected to extraction method mentioned and incubated with different concentrations of urea, β-mercaptoethanol and Tris-HCl buffer, at 25 °C for 4 hours, followed by centrifugation and filtration through a 0.22 μm membrane. The soluble and insoluble fractions before and after solubilization were analyzed using SDS-PAGE 12% gel electrophoresis. It was possible to observe that at 37 °C, the bands were concentrated in the insoluble fraction, that it, predominantly in classic inclusion bodies form. At 20 °C cultivation, the predominant bands were concentrated in the soluble fraction, indicating that the temperature decrease influences the inclusion bodies formation. Lower concentrations of urea, from 1 to 5 M, did not solubilize the inclusion bodies, while higher concentrations, from 6 to 8M, effectively solubilized them. Although, the cultivation carried out at 20 °C did not induce the formation of inclusion bodies, L-asparaginase activity assays did not detect enzymatic activity, but Western Blotting confirmed the presence of the enzyme. Therefore, it will be necessary to purify the solubilized fractions and evaluate different renaturation methodologies for further evaluation of enzymatic activity. Palavras-chave: L-asparaginase, Heterologous expression, fungi, solubilization, inclusion bodies Agência de fomento:FAPDF, FAPESP, CAPES and Univesidade de Brasília | |||||