Cloning of Asparaginase gene, ahrA, from Aspergillus nidulans, and determination of specific enzyme activity using colorimetric methods.

Kyle Smith and Patricia M . Shaffer

Author address: 

Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA, 92110, USA, [email protected], [email protected]


L-Asparaginase is an aminohydrolase that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. The ahrA asparaginase gene (on chromosome VIII) produces a monomeric subunit that has 2 catalytic sites and a single tetrameric interface homology. Now, this gene was purified from previously transformed E.coli, and sequenced to verify the existence of ahrA. The plasmid DNA was used in a PCR reaction with new primers containing BamH1 and HindIII restriction endonuclease sites. The PCR product was ligated into an expression vector (pET-21a(+), Novagen) and was transformed into bacterial strain DH5alpha. A mini-prep and restriction digest was performed to verify the presence of the expected insert into the plasmid. The purified plasmid DNA was transformed into bacterial strain BL21(DE3) that was induced with IPTG to express the enzyme. A 6x His tag allowed for the purification on Ni chelating beads and cleavage from the beads using increasing concentrations of imidizole. The specific enzyme activity (the production of ammonia) was measured by a colorimetric assay using sodium pentacyanonitrosylferrate as the reactive agent. Asparaginase is used as part of the therapy and cure for childhood acute lymphoblastic leukemia (ALL). The success of this research may have medicinal significance. I am grateful to Graduate W omen in Science and San Diego State University Research Foundation for funding and laboratory space.

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Full conference title: 

6th International Aspergillus Meeting
    • Asperfest 6 (2009)