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Cloning, expression, and characterization of a novel thermo-acidophilic l-asparaginase of Pseudomonas aeruginosa CSPS4

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Abstract

In the present investigation, a soil isolate Pseudomonas aeruginosa CSPS4 was used for retrieving the l-asparaginase encoding gene (Asn_PA) of size 1089 bp. The gene was successfully cloned into the pET28a (+) vector and expressed into E. coli BL21(DE3) for characterization of the protein. The recombinant rAsn_PA enzyme was purified by affinity chromatography using Ni-NTA2+ resins. Molecular weight analysis using SDS-PAGE unveiled rAsn_PA as a monomeric protein of molecular weight ~ 35 kDa. On characterization, the recombinant rAsn_PA showed optimum pH and temperature of 6.0 and 60 °C, respectively, along with significant stability at 50–70 °C, along with 50% residual activity at 80 °C after 3 h of incubation. Similarly, the rAsn_PA exhibited asparaginase activity over a broad pH range between 4 and 8. The enzyme was not significantly inhibited in the presence of detergents. The rAsn_PA was grouped into the asparaginase-glutaminase family II due to the glutaminase activity. The purified rAsn_PA showed antitumor activity by exhibiting a cytotoxic effect on three different cell lines, where IC50 of purified rAsn_PA was 2.3 IU, 3.7 IU, and 20.5 IU for HL-60, MOLM-13, and K-562 cell lines, respectively. Thus, recombinant rAsn_PA of P. aeruginosa CSPS4 may also be explored as an antitumor agent after reducing or minimizing the glutaminase activity. Thermo-acidophilic properties of rAsn_PA make it a novel enzyme that needs to be further investigated.

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Data availability

The full-length gene of l-asparaginase reported here can be retrieved using accession no. OR509736 at NCBI.

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Acknowledgements

VK and DV are grateful to Dr. Shilpa Sharma, NSUT, New Delhi for accessing her laboratory for the cloning experiments. AS and CPC are acknowledged for providing facilities to perform cell line experiments at SGPGI, Lucknow. Besides, DV and VK are thankful to Babasaheb Bhimrao Ambedkar University, Lucknow for providing infrastructure and other useful resources.

Funding

There was no financial support received from any funding agency to carry out the work presented here.

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Authors and Affiliations

  1. Department of Environmental Microbiology, School of Earth and Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, 226025, India

    Vinay Kumar & Digvijay Verma

  2. Department of Biological Sciences and Engineering, Netaji Subhas University of Technology, Dwarka, New Delhi, India

    Ravi Kumar & Shilpa Sharma

  3. Department of Applied Sciences and Humanities (Faculty of Technology), University of Delhi, Delhi, India

    Ravi Kumar & Shilpa Sharma

  4. Stem Cell Research Centre, Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

    Arunim Shah & Chandra Prakash Chaturvedi

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  1. Vinay Kumar
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Contributions

DV conceived the idea and coordinated the complete study. VK performed all the experiments. RK and SS assisted in gene cloning. AS and CPC helped in cell line experiments. VK and DV wrote the manuscript. All the authors read, edited, and approved the final version of the manuscript.

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Correspondence to Digvijay Verma.

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Kumar, V., Kumar, R., Sharma, S. et al. Cloning, expression, and characterization of a novel thermo-acidophilic l-asparaginase of Pseudomonas aeruginosa CSPS4. 3 Biotech 14, 54 (2024). https://doi.org/10.1007/s13205-024-03916-9

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