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2030-01-01
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HIGH-YIELD EXTRACTION OF PERIPLASMIC ASPARAGINASE PRODUCED BY RECOMBINANT PICHIA PASTORIS HARBOURING THE SACCHAROMYCES CEREVISIAE ASP3 GENE
Produção de asparaginase
Pichia pastoris
Levedura de enzima periplasmática
Asparaginase production
Periplasmic asparaginase extraction
Yeast periplasmic enzyme
ASP3 gene
Author
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.
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 in Portuguese
Extração de asparaginase periplasmáticaProdução de asparaginase
Pichia pastoris
Levedura de enzima periplasmática
Keywords
Pichia pastorisAsparaginase production
Periplasmic asparaginase extraction
Yeast periplasmic enzyme
ASP3 gene
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