Abstract
L-asparaginase (ASNase) is the principal chemotherapeutic agent against different blood cancers. The risks associated with current clinical preparations demand screening for novel ASNases. Accordingly, the study was conducted to shortlist ASNases having clinically safer profiles from a novel niche, namely, microbes in the gut and hemolymph of apparently healthy Scylla serrata. A four-step strategic approach incorporating the essential requirements for clinically safer profiles was followed. The initial step through plate assay showed five (9.61%) potential ASNase producers. The relative prevalence of ASNase producers was higher in hemolymph (13.33%) than gut (4.5%). The positive isolates were identified as Priestia aryabhattai, Priestia megaterium, Bacillus altitudinis, Shewanella decolorationis, and Chryseomicrobium amylolyticum. Quantitative profiles revealed high ASNase production (114.29 to 287.36 U/mL) without any optimization, with an added advantage of the extracellular production. The second step for substrate specificity studies revealed the absence of L-glutaminase and urease activities in ASNases from C. amylolyticum and P. megaterium, the most desirable properties for safe clinical applications. This is the first report of glutaminase and urease-free ASNase from these two bacteria. The third step ensured type II nature of selected ASNases, the targeted form in clinical applications. The fourth step confirmed the activity and stability in human physiological conditions. Altogether, the results revealed two potential ASNases with clinically compatible profiles.
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Data availability
The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.
Code availability
The sequence data that generated out of the study are submitted in GenBank, NCBI and got assigned with accession codes (MZ918942, MZ916697, MZ914699, MZ914692, and MZ707763).
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Acknowledgements
The authors are grateful to the Head, Marine Biotechnology Division, and the Director, ICAR-Central Marine Fisheries Research Institute, Kochi for providing the necessary facilities to carry out the present investigation. APV acknowledges CSIR-UGC for the research fellowship.
Funding
This work was supported by the KCSTEC funded project “Investigation for L-Asparaginase Free of Clinical Toxicity from Microbial Associations of Marine Crabs” (No. 441/2020/KSCSTE), and ICAR-CMFRI funded project “Health Management in selected finfish and shellfish & bio-prospecting from marine resources” (MBT/HLT/23).
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LVP executed the experimental design, combined the observations, and wrote the manuscript. TGS conceived of the presented idea, supervised the project, and facilitated the experiments. RKJ provided critical feedback and helped in the dissection and hemolymph collection from the crabs. AVN and APV and KTG provided technical support to carry out experiments. NKS helped in procuring the fund and supervising the project.
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All crabs in the present study were handled according to the recommended EU Directive 2010/63/EU for animal experiments and the protocols were approved by ICAR-CMFRI, Kochi-18, India.
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Key Points
• First report on asparaginase from S. decolorationis and C. amylolyticum
• Asparaginases from C. amylolyticum and P. megaterium have clinically safe profiles
• Points out a novel niche for the future asparaginase research
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Lailaja, V.P., Sumithra, T.G., Reshma, K.J. et al. Characterization of novel L-asparaginases having clinically safe profiles from bacteria inhabiting the hemolymph of the crab, Scylla serrata (Forskål, 1775). Folia Microbiol 67, 491–505 (2022). https://doi.org/10.1007/s12223-022-00952-x
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DOI: https://doi.org/10.1007/s12223-022-00952-x