title: Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris.
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.
authors.affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil; Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Luciana
authors.initials: L
authors.lastname: Facchinetti de Castro Girão
authors.affiliation: Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Surza Lucia
authors.initials: SL
authors.lastname: Gonçalves da Rocha
authors.affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ricardo Sposina
authors.initials: RS
authors.lastname: Sobral
authors.affiliation: Oswaldo Cruz Foundation, Biomanguinhos, Laboratory of Macromolecules, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Dinis Ano Bom
authors.affiliation: Oswaldo Cruz Foundation, Farmanguinhos, Institute of Pharmaceutical Technology, 21041-250, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: André Luiz
authors.initials: AL
authors.lastname: Franco Sampaio
authors.affiliation: Oswaldo Cruz Foundation, Biomanguinhos, Laboratory of Macromolecules, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: José
authors.initials: J
authors.lastname: Godinho da Silva
authors.affiliation: Oswaldo Cruz Foundation, Farmanguinhos, Drugs Technology Laboratory, 21041-250, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Maria Antonieta
authors.initials: MA
authors.lastname: Ferrara
authors.affiliation: Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: elba1996@iq.ufrj.br.
authors.forename: Elba
authors.initials: E
authors.lastname: Pinto da Silva Bon
authors.affiliation: Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: jperales@ioc.fiocruz.br.
authors.forename: Jonas
authors.initials: J
authors.lastname: Perales
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D001215:Asparaginase / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D020014:K562 Cells; D008970:Molecular Weight; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics*; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001215:Asparaginase
keywords: Antitumoural drug; Asparaginase; Characterization; Pichia pastoris; Purification