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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 (\n                    <jats:italic>Ea<\/jats:italic>\n                    ) and enthalpy variation (\u0394\n                    <jats:italic>H<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>70\u00baC<\/jats:italic>\n                    <\/jats:sub>\n                    ) 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 (\u0394\n                    <jats:italic>G<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>d<\/jats:italic>\n                    <\/jats:sub>\n                    ) showed higher stabilization by Cys conjugation, while enthalpy (\u0394\n                    <jats:italic>H<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>d<\/jats:italic>\n                    <\/jats:sub>\n                    ) and entropy (\u0394\n                    <jats:italic>S<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>d<\/jats:italic>\n                    <\/jats:sub>\n                    ) evidenced an increase in aggregation upon thermo-inactivation, higher for Cys-PEG-ASNase.\n                  <\/jats:p>","DOI":"10.1007\/s12010-025-05545-1","type":"journal-article","created":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T06:58:25Z","timestamp":1770447505000},"page":"3037-3060","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cysteine Pegylation of a Mutant L-asparaginase Affords Enhanced Activity and Thermostability. A Comparative Study Against N-terminal Conjugation"],"prefix":"10.1007","volume":"198","author":[{"given":"Rafael B.","family":"Ferraro","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guilherme R.","family":"Benevides","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jheniffer","family":"Rabelo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Flaviana","family":"da Silva Chaves","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Grace Veronica","family":"Ruiz-Lara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gustavo","family":"Carretero","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gisele","family":"Monteiro","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adalberto","family":"Pessoa-Junior","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Attilio","family":"Converti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven","family":"Lynham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul F.","family":"Long","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4221-9505","authenticated-orcid":false,"given":"Carlota O.","family":"Rangel-Yagui","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,2,7]]},"reference":[{"key":"5545_CR1","doi-asserted-by":"publisher","first-page":"62","DOI":"10.4103\/0976-500X.184769","volume":"7","author":"RA Egler","year":"2016","unstructured":"Egler, R. 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