Biotechnological enhancement of l-asparaginase production by mutant Aspergillus flavus against MCF-7 breast cancer cells

This study aimed to concentrated on isolating the best fungal strain with the highest l -ASNase activity; strain l -ASNase-4 recorded 296.68 U/mg, was sequenced the genomic region/gene used for molecular identification, identified as Aspergillus flavus AFK3 using PCR and ITS region gene sequencing and submitted into NCBI accession no. OK086056. Sequential mutation induction using Acridin Orange (AO) and Ethidium Bromide (EtBr) were used in successive mutagenesis to increase strain A. flavus AFK3 l -ASNase production. The most successful mutant that produced l -ASNase was A. flavus AO-22, which showed l -ASNase activity two times (622.47 U/mg) over the wild type strain A. flavus AFK3. This study aimed to enhance enzyme production by employing a two-phase optimization strategy. The initial phase employed the One Factor at a Time (OFAT) approach to identify key process parameters (different carbon and nitrogen sources) influencing enzyme activity. OFAT analysis revealed that the best carbon and nitrogen source showed the highest levels of l -ASNase activity in the medium containing glucose and malt extract. In the subsequent phase, Response Surface Methodology (RSM) was used for further optimization, involving 30 experiments to assess the combined effects of multiple factors. The Response Surface Methodology (RSM) was used to identify the ideal conditions for l -ASNase expression. With a pH of 7, 1.5% glucose (a carbon source), 1.5% malt extract (a nitrogen source), and 3 days of incubation, the maximum l -ASNase activity for mutant A. flavus AO-22 was 1923.04 U/mg. Ammonium sulfate precipitation (35–75%), DEAE-cellulose ion exchange chromatography, and Sephadex G100 gel filtration were used to completely purify l -ASNase for the wild type A. flavus AFK3 and its mutant A. flavus AO-22.The purification process raised the purification fold of the purified l -ASNase by 12.79 and 13.09 times, resulting in protein yields of 34.70 and 36.26%; respectively, for the wild type A. flavus AFK3 and its mutant A. flavus AO-22. According to SDS-PAGE analysis, the partially purified l -ASNase in this investigation had a molecular weight of roughly 53 kDa. Under ideal circumstances, a peak specific activity of 8455.83 and 4456.78 U/mg was obtained the purified l -ASNase for the wild type A. flavus AFK3 and its mutant A. flavus AO-22 at 20 min at 30 °C, pH 8.0, using l -asparagine as substrate. Both purified l -ASNase from wild type A. flavus AFK3 and mutant A. flavus AO-22 demonstrated concentration-dependent cytotoxicity in MCF-7 breast cancer cells, as assessed using the MTT assay. Notably, purified l -ASNase from mutant A. flavus AO-22 showed slightly higher cytotoxic potency, with an IC₅₀ of 61.92 µM compared to 64.02 µM for purified l -ASNase from wild type A. flavus AFK3. Overall, both purified l -ASNase from wild type A. flavus AFK3 and mutant A. flavus AO-22 exhibited significant antiproliferative activity, while purified l -ASNase from mutant A. flavus AO-22 showing a marginally stronger effect. According to these results, mutant A. flavus AO-22 seemed to be very future promising effective of l -ASNase a diverse array of clinical uses such as on MCF-7 breast cancer cytotoxicity.