Abstract
Introduction: 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).
Methods: Novel L-asparaginase genes
were identified from a locally isolated
halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified
E. coli strains, OrigamiTM 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.
Results: 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 E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases
(ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an
active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The
Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In
addition, the recombinant ASPase A1 enzyme from Bacillus 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.
Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l
indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.