| Congresso Brasileiro de Microbiologia 2023 | Resumo: 363-1 | ||||
Resumo:L-Asparaginase (L-ASNase) is a therapeutic enzyme used for the treatment of acute lymphoblastic leukemia (ALL), the most common diagnosis of leukemia in children. It is also used in the food industry to reduce acrylamide in baked foods. All the L-ASNases formulations available in the market come from bacteria (Escherichia coli and Dickeya chrysanthemi) and may cause allergy and toxic effects, despite their efficacy in ALL treatment. Furthermore, new sources of L-ASNase are an interesting research topic, aiming for less toxic effects. In industry, fungal L-ASNase is already being successfully employed. Considering fungi cells are more similar to human cells, and L-ASNase has been found in plenty of fungi species, they can be promising for clinical use. In literature, Fungal L-ASNases have already promoted therapeutic features however, fungal yields are low compared to bacterial sources. In the present work, Penicillium sizovae L-ASNase was cloned into an E. coli BL21(DE3) strain to improve P. sizovae L-ASNase (L-ASNasePS) production. Different growth conditions and cell lysis methods were evaluated to maximize enzyme recovery. The expression of the L-ASNasePS was observed by SDS-PAGE and confirmed by Western-Blot in the insoluble fraction, suggesting the presence of inclusion bodies. Different cell lysis sonication protocols were tested. Then, different inducer concentrations at OD600 0.9 and post-induction times were tested, at 37 ºC. Both assays were evaluated by calculating the SDS-PAGE band sizes with the software ImageJ®. The best sonication method found was 10 cycles, 1 min pulse ON 1 min pulse OFF (30% amplitude) (Figure 1). For growth conditions, 0.1 mM of IPTG for 4 h and 0.5 mM of IPTG for 24 h stood out for the enzyme band size-protein content ratio, suggesting high specific activity (Figure 2). These findings pave the way for improving growing techniques and protein preparation for fungal L-ASNase production with a biotechnological approach, using E. coli as a host. Further solubilization and refolding studies are required. Palavras-chave: L-asparaginase, acute lymphoblastic leukemia, protein, fungi, recombinant Agência de fomento:FAPESP e CAPES | |||||