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Volume 26, Issue 4
  • ISSN: 1389-2010
  • E-ISSN: 1873-4316

Abstract

Background

Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia, resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells.

Objective

This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.

Methods

The recombinant L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein's N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized.

Results

The and Vmax values of free rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced L-ASNase thermostability at 37, 45, 50, and 55°C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37°C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form's activity vanished entirely from the rats' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme.

Conclusion

For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged half-life duration, could be considered an alternative to mitigate the high molecular weight of PEGylation's drawbacks.

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Pseudomonas aeruginosa Recombinant L-asparaginase: PEGylation with Low Molecular Weight Polyethylene Glycol, Molecular Dynamics Simulation, In vitro and In vivo Serum half-life and Biochemical Characterization
Current Pharmaceutical Biotechnology 26, 617 (2025); https://doi.org/10.2174/0113892010309260240624072408
/content/journals/cpb/10.2174/0113892010309260240624072408
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  • Article Type:     Research Article
Keyword(s): bio-better; immunogenicity; L-asparaginase; molecular docking; molecular dynamic simulation; PEGylation; pharmacokinetics

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