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-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 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.