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 P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.

Methods: The recombinant P. aeruginosa 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 Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min^-1 and 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 LASNase 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 in vivo 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 in vivo half-life duration, could be considered an alternative to mitigate the high molecular weight of PEGylation's drawbacks.

Keywords: L-asparaginase, PEGylation, immunogenicity, molecular dynamic simulation, molecular docking, pharmacokinetics, bio-better.