Isolation and characterization of fungi producing L-asparaginase with reduced L-glutaminase activity from soil samples

L-asparaginase (L-ASNase), an essential enzyme to treat acute lymphoblastic leukemia (ALL) by depleting leukemia cells' essential nutrient L-asparagine, faces challenges in clinical use due to adverse effects linked to its bacterial origin and L-glutaminase (L-GLNase) co-activity. This study aims to identify fungi capable of producing L-ASNase with reduced L-GLNase co-activity. Among the fungal iolates, isolate JK12 and ChL11 showed high L-ASNase activity (34.04 ± 1.83a U/ml and 30.84 ± 0.53b U/ml, respectively) with reduced L-GLNase co-activity (4.95 ± 0.28c U/ml and 4.80 ± 0.02d U/ml, respectively). Sequencing of the internal transcribed spacer (ITS) region of these isolates identified them as Candida palmioleophila isolate JK12 (≥99% identity with Candida genus) and Trichosporon asahii isolate ChL11 (≥98% identity with Trichosporon genus). Moreover, these isolates exhibited distinct preferences for carbon (C) and nitrogen (N) sources, as well as culture conditions for L-ASNase production. C. palmioleophila isolate JK12 demonstrated the highest L-ASNase production in fructose and yeast extract (67.6 ± 0.04a U/ml and 51.4 ± 0.04a U/ml respectively), following 96 h of incubation at 25°C (43.8 ± 1.22a U/ml, 55.8 ± 0.02a U/ml respectively), with an agitation speed of 100 rpm (59.9 ± 0.04a U/ml). On the other hand, T. asahii isolate ChL11 exhibited maximum L-ASNase production in sucrose and L-asparagine (64.2 ± 0.08a U/ml and 63.6 ± 0.01a U/ml, respectively), after 120 h of incubation at 35°C. The fungal isolates T. asahii isolate ChL11 and C. palmioleophila isolate JK12 have been identified as promising L-ASNase sources of safer therapeutic prospects in cancer therapy due to the reduced GLNase co-activity.