Banca de DEFESA: Samuel Leite Cardoso

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : Samuel Leite Cardoso
DATE: 24/08/2023
TIME: 09:00
LOCAL: Plataforma Zoom
TITLE:

PRODUCTION, PURIFICATION AND CHARACTERIZATION OF FUNGAL LASPARAGINASE IN RECOMBINANT TECHNOLOGY SYSTEMS FROM Pichia Pastoris

KEY WORDS:

L-asparaginase, Pichia pastoris, heterologous proteins, acute lymphoblastic leukemia.

PAGES: 100
BIG AREA: Ciências da Saúde
AREA: Farmácia
SUMMARY:

L-asparaginase is the main therapeutic component in the fight against acute lymphoblastic leukemia. This enzyme catalyzes the hydrolysis reaction of the amino acid L-asparagine into ammonia and aspartic acid and, based on a crucial metabolic difference, is capable of depleting the extracellular levels of the amino acid, making the growth of tumor cells unfeasible. Available in the treatment of acute lymphoblastic leukemia since the 1960s, L-asparaginase is currently produced using recombinant technology in expression systems of heterologous proteins from Escherichia coli and Erwinia chrysanthemi, which directly impacts the treatment and the appearance of several adverse effects, such as fever, increased allergic reactions, hepatotoxicity and changes in thyroid function. Thus, the production of L-asparaginase from eukaryotic organisms could be a strategy to reduce adverse events and increase the therapeutic efficacy of the drug. Therefore, the present work had as main objective the production and purification of recombinant L-asparaginase in heterologous protein production systems of Pichia pastoris. The yeast Pichia pastoris, already recognized for its ability to metabolize methanol, was used with the integration of a coding sequence for L-asparaginase from the fungus Fusarium proliferatum. Total fungal RNA was first extracted and converted to cDNA. After checking the literature and studying the L-asparaginase sequences present in databases, it was possible to construct primers for amplification and recognition of the desired L-asparaginase sequence. After confirmation of the sequence, PPICZαA vectors were obtained from the company InvitrogenTM and first cloned in E. coli. After cloning in E. coli, the vectors were transformed into strains of Pichia pastoris X33, which has a high ability to metabolize methanol through the AOX1 enzyme, mechanisms that are exploited for the production of Lasparaginase. 21 clones were obtained and tested for L-asparaginase production, the highest enzymatic activity found intracellularly was expressed in clone 9 (2.84 IU/g), followed by activities in clones 12, 4.1, 13, 8 and 4. Aiming at a purification process, the enzyme was extracted using a tip sonicator and then subjected to the DEAE Q FF 5 mL ion exchange column. The final results demonstrate a partially purified enzyme that has an optimal pH around 7.0, and Km and Vmax at values of 17.44 mM and 5.35 mM/s-1, respectively. In this way, the integration of L-asparaginase sequences into the eukaryotic DNA of Pichia pastoris and the production of a partially purified functional enzyme can be confirmed.

COMMITTEE MEMBERS:
Externo à Instituição - JOÃO VICENTE BRAGA DE SOUZA - UFAM
Externa à Instituição - LEIA CECILIA DE LIMA FAVARO - EMBRAPA
Externo ao Programa - 1744566 - MAURICIO HOMEM DE MELLO - nullInterna - 1562111 - PEROLA DE OLIVEIRA MAGALHAES DIAS BATISTA
Interna - 1844214 - YRIS MARIA FONSECA BAZZO