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

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.