SummaryDespite many beneficial outcomes of the conventional enzyme replacement therapy (ERT), several limitations such as the high-cost of the treatment and various inadvertent side effects including the occurrence of an immunological response against the infused enzyme and development of resistance to enzymes persist. These issues may limit the desired therapeutic outcomes of a majority of the lysosomal storage diseases (LSDs). Furthermore, the biodistribution of the recombinant enzymes into the target cells within the central nervous system (CNS), bone, cartilage, cornea, and heart still remain unresolved. All these shortcomings necessitate the development of more effective diagnosis and treatment modalities against LSDs. Taken all, maximizing the therapeutic response with minimal undesired side effects might be attainable by the development of targeted enzyme delivery systems (EDSs) as a promising alternative to the LSDs treatments, including different types of mucopolysaccharidoses ( MPSs ) as well as Fabry, Krabbe, Gaucher and Pompe diseases.

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.

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