Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain

Abstract

<b><i>Introduction:</i></b> The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). <b><i>Methods:</i></b> Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant <i>Bacillus</i> strain and the recombinant enzymes were overexpressed in modified <i>E. coli</i> strains, Origami^TM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. <b><i>Results:</i></b> The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by <i>E. coli</i> BL21 strain. The catalytic activity assay on the identified L-asparaginases (<i>ansA1</i> and <i>ansA3</i> genes) from <i>Bacillus</i> sp. SL-1 demonstrated that only <i>ansA1</i> gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The K_cat and K_m values for the purified ASPase A1 enzyme were 23.96^s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from <i>Bacillus</i> sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5-10. <b><i>Conclusion:</i></b> The biochemical properties of the novel ASPase A1 derived from <i>Bacillus</i> sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.