Cysteine Pegylation of a Mutant L-asparaginase Affords Enhanced Activity and Thermostability. A Comparative Study Against N-terminal Conjugation

TL;DR

Researchers improved the L-asparaginase enzyme by pegylating a mutant version at a cysteine site, resulting in better activity and stability compared to N-terminal pegylation.

Contribution

The study introduces cysteine pegylation as a superior method for enhancing ASNase activity and thermostability compared to N-terminal conjugation.

Findings

Cys-PEG-ASNase showed higher specific activity than NT-PEG-ASNase.

Cysteine pegylation provided greater stabilization and increased aggregation upon thermo-inactivation.

Both pegylation methods improved enzyme thermostability and shelf-life.

Abstract

L-asparaginase (ASNase) is one of the most clinically relevant biopharmaceuticals but proteolysis impairs the enzyme half-life. ASNase P40S/S206C was developed to overcome proteolysis and in addition pegylation can be used to improve half-life and thermostability. Here Cys206 and N-terminal residues were explored as pegylation sites for a potential new biobetter. Optimal mono-pegylation of Cys (Cys-PEG-ASNase) provided similar yields compared to N-terminal mono-pegylation (NT-PEG-ASNase) but at lower PEG concentration. Specific activity was higher for Cys-PEG-ASNase than NT-PEG-ASNase. The role of pegylation site in activity was confirmed by activation energy (Ea) and enthalpy variation (ΔH70ºC) of the ASNase-catalysed reaction. Pegylation in both sites increased enzyme thermostability and consequently shelf-life stability. The variation of Gibbs free-energy of enzyme thermo-inactivation (ΔGd) showed higher stabilization by Cys conjugation, while enthalpy (ΔHd) and entropy (ΔSd) evidenced an increase in aggregation upon thermo-inactivation, higher for Cys-PEG-ASNase.

The online version contains supplementary material available at 10.1007/s12010-025-05545-1.

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