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The goal of this work is to study the optimum production of L-ASNase and the influence of oxidized multi-walled carbon nanotubes (oxMWCNTs) on the biological activity of the isolated L-ASNase. Isolation and production of L-ASNase from different screened fungi in the presence of L-asparagine substrate were carried out. The isolated enzyme was identified by molecular18S rRNA analysis. The partial purified L-ASNase having 6.77 purification fold was immobilized onto ox-MWCNTs using physical adsorption technique with maximum yield about 54.4%. In vitro cytotoxicity of L-ASNase against normal fibroblast cell line (BHK-21) was examined relative to the immobilized one using SRB assay. Also, in vivo biological examination using liver and kidney dysfunction biomarkers in the treated mice (AST, ALT, LDH, lipase and α-amylase) was also investigated. Among the ten screened fungi, Aspergillus versicolor was the most potent one with maximum activity about 45.5 U/mL. The results showed that the most appropriate production condition was at asparagine concentration of about 0.04 M and pH 8. The immobilized L-ASNase retained a complete activity (100%) after 30 min at 45OC of incubation relative to that in case of the free one. The toxicity of L-ASNase was significantly decreased up to 50 μg/mL after immobilization onto MWCNTs. Besides, the immobilized L-ASNase exhibited good storage ability and stability at high temperature relative to that in case of the free one.

Production, characterization and immobilization of Aspergillus versicolor L-asparaginase onto multi-walled carbon nanotubes