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<p>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 (<i>Ea</i>) and enthalpy variation (Δ<i>H</i><sub><i>70ºC</i></sub>) 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 (Δ<i>G</i><sub><i>d</i></sub>) showed higher stabilization by Cys conjugation, while enthalpy (Δ<i>H</i><sub><i>d</i></sub>) and entropy (Δ<i>S</i><sub><i>d</i></sub>) evidenced an increase in aggregation upon thermo-inactivation, higher for Cys-PEG-ASNase.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月7日

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  • Rafael B. Ferraro,
  • Guilherme R. Benevides,
  • Jheniffer Rabelo,
  • Flaviana da Silva Chaves,
  • Grace Veronica Ruiz-Lara,
  • Gustavo Carretero,
  • Gisele Monteiro,
  • Adalberto Pessoa-Junior,
  • Attilio Converti,
  • Steven Lynham,
  • Paul F. Long,
  • Carlota O. Rangel-Yagui

摘要

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.