Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/14881
Title: Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris
Authors: Girão, Luciana Facchinetti de Castro
Rocha, Surza Lucia Gonçalves da
Sobral, Ricardo Sposina
Bom, Ana Paula Dinis Ano
Sampaio, André Luiz Franco
Silva, José Godinho da
Ferrara, Maria Antonieta
Bon, Elba Pinto da Silva
Perales, Jonas
Affilliation: Universidade Federal do Rio de Janeiro. Instituto de de Química. Departamento de Bioquímica. 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 de Química. Departamento de Bioquímica. Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Biomanguinhos. Laboratório de Macromoléculas. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Farmanguinhos. Instituto de Tecnologia. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Biomanguinhos. Laboratório de Macromoléculas. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Farmanguinhos. Laboratório de Tecnologia. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de de Química. Departamento de Bioquímica. Laboratório de Tecnologia Enzimática. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.
Abstract: Asparaginase obtained from Escherichia coli and Erwinia chrysanthemi are used to treat acute lymphocytic leukaemia and non-Hodgkin's lymphoma. However, these agents cause severe adverse effects. Saccharomyces cerevisiae asparaginase II, encoded by the ASP3 gene, could be a potential candidate for the formulation of new drugs. This work aimed to purify and characterize the periplasmic asparaginase produced by a recombinant Pichia pastoris strain harbouring the ASP3 gene. The enzyme was purified to homogeneity with an activity recovery of 51.3%. The estimated molecular mass of the enzyme was 136 kDa (under native conditions) and 48.6 kDa and 44.6 kDa (under reducing conditions), suggesting an oligomeric structure. The recombinant asparaginase is apparently non-phosphorylated, and the major difference between the monomers seems to be their degree of glycosylation. The enzyme showed an isoelectric point of 4.5 and maximum activity at 46 °C and pH 7.2, retaining 92% of the activity at 37 °C. Circular dichroism and fluorescence analyses showed that the enzyme structure is predominantly α-helical with the contribution of β-sheet and that it remains stable up to 45 °C and in the pH range of 6-10. In vitro tests indicated that the recombinant asparaginase demonstrated antitumoural activity against K562 leukaemic cells.
Keywords: Asparaginase
Pichia pastoris
Antitumoural drug
Purification
Characterization
keywords: Asparaginase
Drogas antitumorais
Purificação
Caracterização
Pichia pastoris
Issue Date: 2016
Publisher: Elsevier
Citation: GIRÃO, Luciana Facchinetti de Castro; et al. Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris. Protein Expression and Purification, v.120, p. 118-125, 2016.
DOI: 10.1016/j.pep.2015.12.012
ISSN: 1046-5928
Copyright: restricted access
Appears in Collections:Biomanguinhos - Artigos de Periódicos
IOC - Artigos de Periódicos

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