| Author | Ferrara, Maria Antonieta | |
| Author | Severino, Neuza M. Bonomo | |
| Author | Valente, Richard H. | |
| Author | Perales, Jonas | |
| Author | Bon, Elba P. S. | |
| Access date | 2018-04-03T15:23:01Z | |
| Available date | 2018-04-03T15:23:01Z | |
| Document date | 2010 | |
| 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. | pt_BR |
| ISSN | 0141-0229 | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/25585 | |
| Language | eng | pt_BR |
| Publisher | Elsevier | pt_BR |
| Rights | restricted access | |
| Subject in Portuguese | Extração de asparaginase periplasmática | pt_BR |
| Subject in Portuguese | Produção de asparaginase | pt_BR |
| Subject in Portuguese | Pichia pastoris | pt_BR |
| Subject in Portuguese | Levedura de enzima periplasmática | pt_BR |
| Title | High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene | pt_BR |
| Type | Article | |
| DOI | 10.1016/j.enzmictec.2010.05.001 | |
| 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. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Farmaguinhos. Rio de Janeiro, RJ. Brasil. | pt_BR |
| Affilliation | Universidade Federal do Rio de Janeiro. Instituto de Química Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ. Brasil. | pt_BR |
| Affilliation | Universidade Federal do Rio de Janeiro. Instituto de Química Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Subject | Pichia pastoris | pt_BR |
| Subject | Asparaginase production | pt_BR |
| Subject | Periplasmic asparaginase extraction | pt_BR |
| Subject | Yeast periplasmic enzyme | pt_BR |
| Subject | ASP3 gene | pt_BR |
| e-ISSN | 1879-0909 | |
| Embargo date | 2030-01-01 | |
