Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase |
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| Authors: | Hesham Saeed Hadeer Soudan Amany El-Sharkawy Aida Farag Amira Embaby Farid Ataya |
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| Institution: | 1.Department of Biotechnology, Institute of Graduate Studies and Research,Alexandria University,Alexandria,Egypt;2.Marine Biotechnology and Natural Products Extract Laboratory,National Institute of Oceanography and Fisheries (NIOF),Alexandria,Egypt;3.Biochemistry Department, College of Science,King Saud University,Riyadh,Kingdom of Saudi Arabia;4.National Research Centre,Giza,Egypt |
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| Abstract: | Recombinant l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg2+ and inhibited by Zn2+ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately. |
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