Antitumor activity of urease-free l-asparaginase with low glutaminase coactivity produced by marine-derived Aspergillus flavus

Abstract

l-asparaginase, which has a high therapeutic effect in various cancer diseases, hydrolyzes l-asparagine to aspartate and ammonia and is mainly used in treating Acute Lymphoblastic Leukaemia. This study aims to investigate the l-asparaginase production potential of marine filamentous fungi and the partial purification and characterization of the l-asparaginase. The rapid plate screening method was used to measure the l-asparaginase activity of 94 marine fungi. l-asparaginase producers were then screened for glutaminase and urease activity. The selected extracellular l-asparaginase producer, Aspergillus flavus MF10, showed 2.82 U/mL l-asparaginase activity in small-scale fermentation. l-asparaginase was concentrated by ultrafiltration followed by acetone precipitation. The specific activity of the enzyme, purified 4,8 fold by partial purification, was determined as 259 U/mg. There were no urease and glutaminase activities in the fermentation broth, but low glutaminase activity (1.27 U/mL) was detected in the concentrated fermentation broth. The optimum pH and temperature of the l-asparaginase were 8.0 and 40 °C, respectively. l-asparaginase was stable at pH 7.0–8.0 and can maintain its activity at 70% at 40 °C after 1 h. While the enzyme decreased the viability of SH-SY5Y (neuroblastoma) to 43% and LNCap (prostate cancer) to 61%, it did not show cytotoxic effects in healthy cell line L929 (fibroblast). It was also determined that l-asparaginase showed low cytotoxicity in HaCaT (keratinocyte) cells. In conclusion, it was shown that urease-free l-asparaginase with low glutaminase coactivity, produced from marine-derived A. flavus MF10, has the potential for medical use.