| Congresso Brasileiro de Microbiologia 2023 | Resumo: 662-1 | ||||
Resumo:L-asparaginase is the enzyme responsible for the hydrolysis of the amino acid L-asparagine into aspartic acid and ammonia. This enzyme is used for the treatment of Acute Lymphoblastic Leukemia (ALL) and plays an important role in the induction therapy during the initial phase of treatment, aiming to reduce the number of leukemic cells. The enzyme hydrolyzes L-asparagine present in the blood, preventing this amino acid from being used as a nutrient by tumor cells. The enzyme used in ALL therapy is from a bacterial source, and is associated with significant adverse reactions reported by patients. It is known that filamentous fungi can express this protein, making the study of these sources interesting since they are eukaryotic microorganisms. In this regard, studying the production of this enzyme by cloning and expressing the L-asparaginase gene from Fusarium proliferatum in Escherichia coli proves to be promising. The objective of this study was to analyze variables related to the induction of the recombinant enzyme, including the optimal induction temperature, time after induction and concentration of the inducer IPTG. The L-asparaginase gene obtained from Fusarium proliferatum was sequenced and inserted into the pET-28a(+) vector for transformation into two strains of BL21 (DE3) Escherichia coli, one native and one mutated. An inoculum was prepared from these strains in a 50 mL Falcon tube containing 10 mL of LB medium and 50 μg/mL of kanamycin. The inoculum was then incubated at 200 rpm and 37 ºC for 2 hours and 30 minutes. After this period, the expression was induced using IPTG, varying the concentration of the inducer as well as different temperatures and post-induction times, as described in the table below. The samples were sonicated and subjected to analysis by SDS-PAGE. The activity of L-asparaginase was measured using the Nessler method, a colorimetric assay commonly used to measure the activity of enzymes that produce ammonia. The SDS-PAGE analysis revealed the presence of a prominent band, indicating that the highest expression of both the native and mutated enzymes occurred after 12
hours of induction at 20 °C with 0.5 mM IPTG. Additionally, it was noted that under these conditions, the enzyme predominantly remained in the soluble fraction. Besides, the mutated enzyme, when cultivated at 37 °C with 0.5 mM IPTG, exhibited significant bands in the insoluble fraction and the absence of protein in the soluble fraction. Using the Nessler method, the native enzyme displayed an activity of 0.105 U/mL, while the mutated enzyme showed activity in the range of 0.017 U/mL, both cultivated at 20 °C. Meanwhile, the enzymatic activity of the insoluble fractions obtained from the cultivation of E. coli BL21(DE3) at 37 °C was not relevant. As the native enzyme exhibited L-asparaginase activity in the soluble fraction, it was concentrated 10-fold using a 30 kDa centrifugal concentrator under the conditions of centrifugation at 8000 x g, 4 °C for 15 minutes. The activity assay was then repeated, resulting in an activity of 0.628 U/mL. At a temperature of 37 °C, it was observed that the enzyme did not exhibit activity in any of the analyzed fractions. However, at a temperature of 20 °C, even at low levels, enzymatic activity was detected. The Nessler assay provided evidence that temperature variation influences the formation of inclusion bodies and, consequently, affects enzymatic activity. Palavras-chave: L-asparaginase, Leukemia, Heterologous Expression Agência de fomento:Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq | |||||