Abstract
With the emergence of multiple side effects on the usage of commercial L-asparaginase formulations, keen interest is provoked to investigate new sources of L-asparaginases that possess antileukemic properties with minimal side effects. The present study reports the cost-effective bench-scale production, homogeneity purification and apoptosis induction potential of a new L-asparaginase preparation from Bacillus indicus against human leukemia cells. The enzyme is highly specific toward the natural substrate L-asparagine. The study initiated with the enzyme production using cost-effective substrates in which a 3.28-fold enhancement of enzyme activity was achieved in comparison with an unoptimized medium using the central composite experimental design approach. The scale-up of the process in a 3.7-L batch bioreactor resulted in 16.42 ± 0.17 IU/mL of L-asparaginase activity in 24 h. The crude extracellular enzyme was purified to homogeneity using anion exchange chromatography followed by gel filtration chromatography. A single band of approximately 35 kDa molecular weight was obtained on SDS-PAGE, while native PAGE analysis confirmed it to be a tetramer of four identical subunits. The circular dichroism spectroscopic study revealed the α + β mixed type of secondary structure with 38.7% α-helices and 27.4% β pleated sheets. The antitumor toxicity exhibited on the MOLT-4 leukemia cells by the new L-asparaginase was revealed using the MTT assay and acridine orange/propidium iodide dual staining for live/dead cells. The flow cytometry analysis established the potential of the purified L-asparaginase to induce the apoptotic cell death mechanism in MOLT-4 leukemia cells. Conclusively, the L-asparaginase of Bacillus indicus is a highly promising candidate that can be introduced as a new enzyme therapeutic against various leukemia disorders.
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We thank School of Biochemical engineering, Indian Institute of Technology BHU Varanasi, India, for providing us the infrastructure and facilities for conducting research work.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by DS and AM. The first draft of the manuscript was written by DS, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Sharma, D., Mishra, A. Apoptosis induction in leukemic cells by L-asparaginase preparation from Bacillus indicus: bench-scale production, purification and therapeutic application. 3 Biotech 13, 21 (2023). https://doi.org/10.1007/s13205-022-03440-8
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DOI: https://doi.org/10.1007/s13205-022-03440-8