Banca de DEFESA: THAIS PAIVA PORTO DE SOUZA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : THAIS PAIVA PORTO DE SOUZA
DATE: 09/04/2021
TIME: 08:30
LOCAL: Remoto via plataforma Google Meet
TITLE:

Expression of Aspergillus nidulans phytase in cells of Komagataella phaffii (Pichia pastoris) and analysis of its stability at different pHs by molecular dynamics

KEY WORDS:

phytase, phytate, komagataella pastoris, heterologous expression

PAGES: 164
BIG AREA: Multidisciplinar
AREA: Biotecnologia
SUMMARY:

Phytate or phytic acid (Inositol hexaphosphate: C6H18O24P6 - IP6) is present in plants, especially in cereals such as soybeans, corn, wheat, oats and legumes. Currently, phytate is considered an anti-nutritional factor because it binds to metals and minerals, such as iron, zinc, copper and mainly phosphorus, reducing its absorption during digestion in monogastric animals or even in humans. Phytases are enzymes capable of degrading phytic acid and have a wide range of applications in industries, such as animal feed supplementation in order to improve digestibility through the release of nutrients such as phosphorus, present in the form of phytate. This work concerns the construction of a lineage of the yeast Komagataella phaffii (Pichia pastoris) as an expression system for the production of the phytase enzyme. Therefore, a screening of phytases with relevant biotechnological characteristics aiming at a rational design of the proposal was carried out. Filamentous fungi phytases are currently promising, as they have desirable biochemical properties for application in the animal industry, such as thermostability and stability in different pH profiles. Therefore, the phytase of Aspergillus nidulans was chosen for production and the K. phaffii GS115 strain was used as a system for expression. Bioinformatic analyzes (in silico analyzes) of the gene sequence were performed to optimize the sequences obtained from GenBank. The plasmid pPICZα-B containing the phytase ORF was synthesized and cloned in vitro with codons optimized for expression in K. paffi. From this, the plasmid was amplified in Escherichia coli strains, later extracted by medium scale preparation and detected by PCR. The vector was linearized with the restriction enzyme Sac I and cloned into K. phaffii GS115. The expression was carried out with 0.5% methanol at different induction times, so that the protein bands were analyzed using SDS-PAGE polyacrylamide gel. The phenotypes of the obtained clones were determined for methanol metabolism. In addition, it was possible to select the clone with the highest enzyme activity against the hydrolysis of sodium phytate. Molecular and metadinamic dynamics studies were also carried out to check the enzymatic stability in different ranges of hydrogen potential (pH), in relation to the presence and absence of protein glycosylation.

BANKING MEMBERS:
Presidente - 1367304 - ALEXSANDRO SOBREIRA GALDINO
Externa à Instituição - LUCIANA DE PAULA NAVES - UFLA
Externo à Instituição - MARINA QUÁDRIO RAPOSO BRANCO RODRIGUES - UNIFAL-MG
Externo à Instituição - ADRIANO GUIMARAES PARREIRA - UEMG
Externo à Instituição - MICHEL RODRIGO ZAMBRANO PASSARINI - UNILA
Externa à Instituição - SABRINA DE AZEVEDO SILVEIRA - UFV