Banca de DEFESA: Marina Borges Guimarães

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : Marina Borges Guimarães
DATE: 26/11/2024
TIME: 14:00
LOCAL: Plataforma Virtual - ConferênciaWeb
TITLE:

L-ASPARAGINASE FROM Penicillium sizovae, A FILAMENTOUS FUNGUS FROM CERRADO

KEY WORDS:

L-asparaginase; filamentous fungi; heterologous expression; acute lymphoid leucemia; aqueous biphasic system.

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

The enzyme L-asparaginase (L-ASNase) was the first therapeutic enzyme discovered and treats leukemia. Its mechanism of action consists of catalyzing the hydrolysis of L-asparagine into aspartic acid and ammonia. Despite being the first line of treatment for ALL, there is still no production of LASNase in Brazil. Furthermore, all approved and commercially available L-ASNase formulations are of bacterial origin (from native and pegylated Escherichia coli, and Dickeya dadantii); however, these formulations can cause toxicity and hypersensitivity in some patients. Aiming the production of an LASNase with fewer possible adverse effects and higher yield, this work investigates the L-ASNase from the fungus Penicillium sizovae, isolated from the soil of the Cerrado. Two approaches are evaluated: the native enzyme and the enzyme heterologously expressed in the prokaryotic system E. coli BL21(DE3). The production of the native enzyme is optimized considering low-cost conditions and less environmental impact. After screening the culture medium, extraction methods, and solvent used, purification was performed in a two-phase aqueous polymer/salt system, with the PEG2000/phosphate buffer system presenting the highest purification factor (1.63). Ion exchange chromatography was recovery yield up to 33.66%. The enzyme expressed in E. coli had its cloning and expression confirmed by PCR, SDS-PAGE, and Western Blot. The presence of the enzyme was observed mainly in the insoluble fractions, suggesting the formation of inclusion bodies (IC). To identify the best cultivation conditions in both the soluble and insoluble fractions, an expression optimization was performed considering inducer concentrations, post-induction time, and temperature. Still expressed in CI, solubilization tests were performed with guanidine hydrochloride, and then an experimental matrix to find the best additives for its refolding in the original conformation. The tetrahedral conformation was confirmed with native-PAGE; however, the enzyme did not have activity. New studies should be carried out to find the ideal condition for this recombinant L-ASNase present enzymatic activity.

COMMITTEE MEMBERS:
Externa à Instituição - VALÉRIA DE CARVALHO SANTOS EBINUMA - UNESP
Externa à Instituição - MÔNICA CARAMEZ TRICHES DAMASO - EMBRAPA
Interna - 3231108 - PAULA MONTEIRO DE SOUZA
Presidente - 1562111 - PEROLA DE OLIVEIRA MAGALHAES DIAS BATISTA
Externo à Instituição - SAMUEL LEITE CARDOSO - UPIS