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Increased glycosylated L-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches
- Source :
- Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual), Universidade de São Paulo (USP), instacron:USP
- Publication Year :
- 2021
-
Abstract
- Currently, only the biopharmaceutical l -asparaginase (ASNase), a chemotherapy agent, from bacteria is approved for clinical use. Although it is efficient, it causes severe side effects due to its origin. Therefore, new sources are investigated to reduce immunogenicity. Moreover, it is urgent to enhance protein stability, folding and kinetics with post-translational modifications, such as glycosylation. Aiming at these attributes, this study focused on the production of a glycosylated l -asparaginase of Dickeya chrysanthemi expressed by Pichia pastoris Glycoswitch®. Producer strains of Pichia, one with auxotrophy for histidine and another prototrophic were compared by using a complex medium in flasks and a synthetic medium in a bioreactor. The prototrophic strain showed higher productivity than the auxotrophic in both scales. Following, two induction strategies with the prototrophic strain were executed: methanol pulses and a DO-stat. The latter resulted in 2-fold more maximum volumetric activity. Lastly, the glycosylation analysis of the final product showed that the site Asn170, which is associated with allergies in patients, was glycosylated, thus, reducing potential immunogenic effects. Therefore, this study showed that the prototrophic strain was the most suitable l -asparaginase producer and that methanol–oxygen control in bioreactor enhanced the production of a glycosylated ASNase.
- Subjects :
- 0106 biological sciences
0303 health sciences
Asparaginase
Environmental Engineering
Glycosylation
biology
ANTINEOPLÁSICOS
Auxotrophy
Biomedical Engineering
Bioengineering
biology.organism_classification
01 natural sciences
Pichia pastoris
carbohydrates (lipids)
03 medical and health sciences
chemistry.chemical_compound
Biochemistry
chemistry
010608 biotechnology
Bioreactor
Bacteria
Histidine
030304 developmental biology
Biotechnology
Pichia
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual), Universidade de São Paulo (USP), instacron:USP
- Accession number :
- edsair.doi.dedup.....8ff3e209948b6b534d51fadf0a2cbae0