Banca de QUALIFICAÇÃO: Marina Borges Guimarães

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
STUDENT : Marina Borges Guimarães
DATE: 01/11/2022
TIME: 14:00
LOCAL: Plataform Conferência Web UnB
TITLE:

HETEROLOGOUS EXPRESSION OF Penicillium sizovae L-ASPARAGINASE in Escherichia coli BL21 (DE3)

KEY WORDS:

L-asparaginase; fungi; heterologous expression; acute lymphoid leukemia

PAGES: 100
BIG AREA: Ciências da Saúde
AREA: Fisioterapia e Terapia Ocupacional
SUMMARY:

L-asparaginase (L-ASNase) was the first therapeutic enzyme discovered and is part of leukemia treatment. Its action mechanism consists of catalyzing the hydrolyses of Lasparagine into acid aspartic and ammonia. Besides being the first treatment line for ALL, there is no L-ASNase production in Brazil. Furthermore, all L-ASNase approved and available in the market formulations are from a bacterial source (from Escherichia coli native and peguilated, and from Erwinia chrysantemi), however, these formulations cause toxicity and hypersensibility in the patients. L-ASNases from fungi have fewer adverse effects, however, their productivity is low. Aiming the production of an L-ASNase with fewer adverse effects and higher yield, this work has the cloning and expression of the L-ASNase gene from Penicillium sizovae in E. coli BL21 (DE3). The cloning was confirmed by PCR and Western Blot. Cell lysis was made by sonication, and soluble and insoluble fractions were evaluated by band intensity of SDS-PAGE. It was observed that the enzyme was present only in the insoluble fractions, suggesting the presence of inclusion bodies (IC). To identify better culture conditions both in soluble and insoluble fractions, a screening was made with different IPTG concentrations (0.1 mM; 0.5 mM, and 1 mM), time post-induction (2 h, 4 h, 8h, 12 h, 20 h, and 24 h), and induction temperature (20 ºC and 37 ºC). After the selection of cultures by band intensity in SDS-PAGE, L-ASNase activity was obtained by AHA method in the soluble fraction of cultures at 20 ºC, the highest activity was 0.02 U/mL (0.5 mM of IPTG and 24 h pos-induction). The enzyme activity was also tested in the biomass of these cultures and the highest values were in the conditions of 0.5 mM of IPTG, 24 h post-induction (2.5 U/gcell), and 0.5 mM of IPTG, 4 h post-induction (1.9 U/gcell). In the selected cultures at 37 ºC, there was no L-ASNase activity in both soluble and insoluble fractions, indicating CI formation. Solubilization tests with urea in concentrations from 2 – 8 M were made to identify urea molarity able to solubilize IC from each culture. Still so, refolding tests are required to return the enzyme to its original and active conformation.

BANKING MEMBERS:
Externo ao Programa - 2122696 - EDIVALDO XIMENES FERREIRA FILHO
Externa ao Programa - 2222088 - ELIANE FERREIRA NORONHA
Externa ao Programa - 1529483 - MARIA DE FATIMA BORIN
Presidente - 1562111 - PEROLA DE OLIVEIRA MAGALHAES DIAS BATISTA
Externo ao Programa - 1661623 - RICARDO HENRIQUE KRUGER