Production of the Anti-Leukemic Therapeutic Enzyme, L-Asparaginase, by A Brackish Sediment Strain of Aspergillus candidus

Aspergillus candidus strain IR-A4 was isolated from the brackish sediment of Itu River, Akwa Ibom State, Nigeria on Czapex-Dox yeast-extract agar and screened for L-asparaginase production by the rapid plate technique and in submerged fermentation. Protein was detected in cell-free fermentation broth by the Bradford method and confirmed as L-asparaginase by rapid development of pink colour on asparagine-minimal medium. The protein was partially purified by (NH4)2SO4 precipitation and dialysis against Tris-HCl buffer. L-asparaginase activity, evaluated by the colorimetric Nesslerization method, was 1282 ± 70.5 U with a specific activity of 17.26 U/mg. The enzyme demonstrated very minimal glutaminase specific activity of 0.03 U/mg with a 352-fold lower specific activity relative to L-asparaginase activity suggesting near-free allergic reactions in the course of therapy. The anti-leukemic activity of the enzyme, demonstrated by in vitro cytotoxicity assay using HL-60 cell lines, showed that 87.98% of the dialyzed fraction of the enzyme could destroy 50% (IC50) of leukemic cells. There was 97% association, ω2, between fermentor size and enzyme activity suggesting great potential for large-scale fermentative production of the therapeutic enzyme. Optimal conditions for enzyme activity were set at (i) Substrate concentration, 220-260 mM (ii) pH 8-9 and (iii) Temperature, 35-45ºC. The mould is recommended for large-scale production of low-glutaminase activity L-asparaginase for treatment of lymphoblastic leukemia patients.