High-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene

dc.creatorFerrara, Maria Antonieta
dc.creatorSeverino, Neuza M. Bonomo
dc.creatorValente, Richard H.
dc.creatorPerales, Jonas
dc.creatorBon, Elba P. S.
dc.date2018-04-03T15:23:01Z
dc.date2018-04-03T15:23:01Z
dc.date2010
dc.date.accessioned2023-09-26T20:16:46Z
dc.date.available2023-09-26T20:16:46Z
dc.identifierFERRARA, 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.
dc.identifier0141-0229
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/25585
dc.identifier10.1016/j.enzmictec.2010.05.001
dc.identifier1879-0909
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8853181
dc.descriptionThe 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.
dc.description2030-01-01
dc.formatapplication/pdf
dc.languageeng
dc.publisherElsevier
dc.rightsrestricted access
dc.subjectExtração de asparaginase periplasmática
dc.subjectProdução de asparaginase
dc.subjectPichia pastoris
dc.subjectLevedura de enzima periplasmática
dc.subjectPichia pastoris
dc.subjectAsparaginase production
dc.subjectPeriplasmic asparaginase extraction
dc.subjectYeast periplasmic enzyme
dc.subjectASP3 gene
dc.titleHigh-yield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3 gene
dc.typeArticle


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