Immobilization of L-asparaginase on oxidized bacterial cellulose to enhance stability and cytotoxic activity

L-asparaginase (L-ASNase) is a key therapeutic enzyme used to treat acute lymphoblastic leukemia (ALL). However, its use against solid tumors cells is limited due to its instability, rapid inactivation, and resistance mechanisms; thus, developing novel carriers that can improve L-ASNase stability while preserving its antitumor activity is crucial. In this study, oxidized BC (OBC) films were used for the first time as covalent carriers for L-ASNase from Erwinia carotovora (EwA) to achieve prolonged action against cancer cells. Oxidizing BC films with sodium periodate (NaIO₄) or 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) introduces reactive aldehyde or carboxyl groups, which enables stable covalent attachment of EwA. Immobilizing EwA on TEMPO-OBC and NaIO₄-OBC films significantly increased the enzyme's thermal stability, raising its inactivation temperatures from 40 °C for the free enzyme to 50 °C for the TEMPO-oxidized BC and 60 °C for the NaIO₄-oxidized BC. The pH range of activity for the immobilized L-ASNase was wider (pH 6–11) than that of the free enzyme (pH 8–9). Furthermore, EwA immobilized on NaIO₄-OBC exhibited pronounced resistance to trypsin and urea, retaining 100 % activity after eight reuse cycles. Cytotoxicity assays demonstrated that EwA immobilized on NaIO₄-OBC effectively induced cell death in solid tumor cell lines that were previously insensitive to the free enzyme. This was due to its enhanced stability and sustained activity. The decrease in cell viability was associated with L-asparagine depletion rather than L-glutamine depletion in the cell culture medium. These results suggest that covalently immobilizing EwA on NaIO₄-OBC is a promising way to improve L-ASNase stability and expand its use in treating solid tumors. © 2025 Elsevier B.V.