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Characterization, Anti-proliferative Activity, and Bench-Scale Production of Novel pH-Stable and Thermotolerant L-Asparaginase from Bacillus licheniformis PPD37

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Abstract

Bacterial L-asparaginase (LA) is a chemotherapeutic drug that has remained mainstay of cancer treatment for several decades. LA has been extensively used worldwide for the treatment of acute lymphoblastic leukemia (ALL). A halotolerant bacterial strain Bacillus licheniformis sp. isolated from marine environment was used for LA production. The enzyme produced was subjected to purification and physico-chemical characterisation. Purified LA was thermotolerant and demonstrated more than 90% enzyme activity after 1 h of incubation at 80 °C. LA has also proved to be resistant against pH gradient and retained activity at pH ranging from 3.0 to 10. The enzyme also had high salinity tolerance with 90% LA activity at 10% NaCl concentration. Detergents like Triton X-100 and Tween-80 were observed to inhibit LA activity while more than 70% catalytic activity was maintained in the presence of metals. Electrophoretic analysis revealed that LA is a heterodimer (~ 63 and ~ 65 kDa) and has molecular mass of around 130 kDa in native form. The kinetic parameters of LA were tested with LA having low Km value of 1.518 µM and Vmax value of 6.94 µM/min/mL. Purified LA has also exhibited noteworthy antiproliferative activity against cancer cell lines—HeLa, SiHa, A549, and SH-SY-5Y. In addition, bench-scale LA production was conducted in a 5-L bioreactor using moringa leaves as cost-effective substrate.

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The datasets supporting the conclusions of this article are included within the article and its additional files.

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Acknowledgements

The authors are grateful to Indrashil University for providing financial support and infrastructural facilities to carry out present research work. AP would like to acknowledge Education Department, Government Gujarat, for Scheme of Developing High Quality Research (SHODH)-2021 fellowship (Ref No. 202001420005).

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Authors and Affiliations

  1. Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, 382740, India

    Payal Patel, Ajay Patel, Bharti Dave & Haren Gosai

  2. Immunology Lab, Indian Institute of Advanced Research (IIAR), Gandhinagar, Gujarat, India

    Reena Agarwal-Rajput

  3. Department of Biochemistry & Forensic Science, Gujarat University, Ahmedabad, Gujarat, India

    Rakesh Rawal

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  1. Payal Patel
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  2. Ajay Patel
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Contributions

Payal Patel: literature review, data curation, formal analysis, methodology, writing – original draft. Ajay Patel: data curation, methodology. Reena Agarwal-Rajput: resources, data curation, methodology. Rakesh Rawal: resources, data curation, methodology. Bharti Dave: writing- review and editing. Haren Gosai: conceptualization, data curation, methodology, supervision, writing – review &editing, validation, and resources.

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Correspondence to Haren Gosai.

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Highlights

• LA from marine-derived Bacillus licheniformis had specific activity of 7707 U/mg.

• Purified LA proved to be thermotolerant and resistant to temperature up to 80 °C.

• pH stability was exhibited by purified LA at pH 3.0–10.

• LA was resistant to high salinity concentration of 3–16%.

• IC50 values of less than 0.2 U/mL was observed against cancer cell lines.

• Antibacterial activity against Vibrio cholerae and Enterococcus faecalis was detected.

• Economical bench-scale production of LA using moringa leaves.

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Patel, P., Patel, A., Agarwal-Rajput, R. et al. Characterization, Anti-proliferative Activity, and Bench-Scale Production of Novel pH-Stable and Thermotolerant L-Asparaginase from Bacillus licheniformis PPD37. Appl Biochem Biotechnol 195, 3122–3141 (2023). https://doi.org/10.1007/s12010-022-04281-0

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  • DOI: https://doi.org/10.1007/s12010-022-04281-0

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