Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/25585
Title: High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene
Authors: Ferrara, Maria Antonieta
Severino, Neuza M. Bonomo
Valente, Richard H.
Perales, Jonas
Bon, Elba P. S.
Affilliation: Fundação Oswaldo Cruz. Farmaguinhos. Rio de Janeiro, RJ. Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Química Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Química Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil.
Abstract: The enzyme asparaginase is used for the treatment of haematopoietic diseases, such as acute lymphoblastic leukaemia and non-Hodgkin lymphomas. The extraction of the periplasmic asparaginase produced in high levels by a recombinant Pichia pastoris strain harbouring the Saccharomyces cerevisiae ASP3 gene was studied. We submitted the yeast cells to freeze–thaw cycles, ethanol treatment and alkaline extraction in the presence and absence of cysteine. The use of six freeze–thaw cycles, followed by extraction with 20mM potassium phosphate buffer pH 7.0 for 20 h, resulted in 85% enzyme recovery whereas the alkaline extraction using 500mM potassium phosphate at pH 11.5 in the presence of 10mM cysteine allowed 100% enzyme recovery. The protein and asparaginase concentrations in the crude extract for the alkaline cysteine treatment (1220mgL−1 protein; 19,134UL−1 asparaginase) were higher than those observed for the freeze–thaw procedure (840mgL−1; 13,274UL−1). The activities of the two aforementioned asparaginase crude preparations were stable upon storage at−18 ◦C for several months. SDS-PAGE analysis of the two extracts displayed two major protein bands from each extraction protocol, that were both identified as asparaginase II from S. cerevisiae by mass spectrometric analyses.
Keywords: Pichia pastoris
Asparaginase production
Periplasmic asparaginase extraction
Yeast periplasmic enzyme
ASP3 gene
keywords: Extração de asparaginase periplasmática
Produção de asparaginase
Pichia pastoris
Levedura de enzima periplasmática
Issue Date: 2010
Publisher: Elsevier
Citation: FERRARA, Maria Antonieta; et al. High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene. Enzyme and Microbial Technology, v.47, p.71–76, 2010.
DOI: 10.1016/j.enzmictec.2010.05.001
ISSN: 0141-0229
Copyright: restricted access
Appears in Collections:Farmanguinhos - Artigos de Periódicos
IOC - Artigos de Periódicos

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