Enzymatic characterization of Q59 l-ASP mutants. (A) Coomassie blue–stained SDS-PAGE showing expression of l-ASP WT and Q59 mutants. The expression vector was transformed into E coli Bl-21 strain, and 20 µL of culture supernatant was analyzed by SDS-PAGE. The empty expression vector (Ctrl) and T89V (inactive mutant) served as negative controls for assays of enzyme activity in panels B and C. (B) Asparaginase activity of Q59 mutants by colorimetric assay. (C) Glutaminase activity of Q59 mutants by colorimetric assay. (D) Asparaginase-specific activity of purified Q59 mutants by the colorimetric assay. (E) Glutaminase-specific activity of purified Q59 mutants by the colorimetric assay. (F) Ratio of glutaminase- and asparaginase-specific activities for purified l-ASP mutants. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.