Cloning, Expression, and Characterization of l-Asparaginase from a Newly Isolated Bacillus subtilis B11–06

This study focused on the cloning, overexpression, and characterization of the gene encoding l-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11–06. The recombinant enzyme showed high thermostability and low affinity to l-glutamine. The ansZ gene, encoding a putative l-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11–06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0–9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The Km for l-asparagine was 0.43 mM, and the Vmax was 77.51 μM/min. Results of this study also revealed the...