Abstract
L-Asparaginase II from Erwinia carotovora may represent an important alternative therapy in the treatment of acute childhood lymphoblastic leukaemia, despite its promising lower glutaminase activity than Escherichia coli and Erwinia chrysanthemi L-asparaginases II, currently used in treatment of this disease. Here we describe cloning, expression, purification and determination of steady-state kinetic parameters for E. carotovora L-asparaginase II: with (AspSP) and without the signal peptide (AspMP). AspMP was purified to homogeneity by a single-step protocol with 91% yield, and AspSP by a two-step protocol with 28% yield. In addition, both enzymes presented similar high specific activities: 208.1 and 237.6 U mg-1, respectively. The Km and kcat values showed that AspMP has lower glutaminase activity than AspSP. Moreover AspMP is produced by a simpler purification protocol, and at higher yield. This process can be amenable to large scale production and be of interest to researchers and biopharmaceutical companies
Highlights
L-asparaginase enzymes (L-Asparagine amidohydrolase; EC 3.5.1.1) catalyze the hydrolysis of L-asparagine (L-Asn) to Laspartate (L-Asp) and ammonia (NH3), and to a lesser extent the hydrolysis of L-glutamine (L-Gln) to L-glutamate (L-Glu)
The main side effects are liver dysfunction, pancreatitis, diabetes, leucopoenia, neurological seizures, and coagulation abnormalities that may lead to intracranial thrombosis or hemorrhage (Duval et al, 2002; Oettgen et al, 1970). Another limiting factor of E. coli L-asparaginase II is the development of hypersensitivity, which ranges from mild allergic reactions to anaphylactic shock (Schwartz et al, 1966)
Heterologous expression of E. carotovora AspMP in E. coli BL21(DE3) host cells could be achieved in the presence and absence of isopropyl β-Dthiogalactopyranoside (IPTG) induction as shown by a strong protein band of approximately 39 kDa as determined by SDS-PAGE analysis of the soluble fraction (Figure 1)
Summary
L-asparaginase enzymes (L-Asparagine amidohydrolase; EC 3.5.1.1) catalyze the hydrolysis of L-asparagine (L-Asn) to Laspartate (L-Asp) and ammonia (NH3), and to a lesser extent the hydrolysis of L-glutamine (L-Gln) to L-glutamate (L-Glu). Type I L-asparaginases are expressed constitutively in the cytoplasm and catalyze the hydrolysis of both L-Asn and L-Gln, whereas type II Lasparaginases are expressed under anaerobic conditions in the periplasmic space of the bacterial membranes and display higher specificity for L-Asn hydrolysis (Campbell et al, 1967; Cedar and Schwartz, 1968). The main side effects are liver dysfunction, pancreatitis, diabetes, leucopoenia, neurological seizures, and coagulation abnormalities that may lead to intracranial thrombosis or hemorrhage (Duval et al, 2002; Oettgen et al, 1970). Another limiting factor of E. coli L-asparaginase II is the development of hypersensitivity, which ranges from mild allergic reactions to anaphylactic shock (Schwartz et al, 1966). Because L-asparaginases II from E. coli and Erwinia possess different immunological specificities they offer an important alternative therapy if a patient becomes hypersensitive to one of these enzymes (Lee et al, 1989)
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