Immobilization of Novel l -asparaginase Variants on Functionalized Multiwalled Carbon Nanotubes Improves Stability, Resistance to Proteolytic Degradation, and Prolonged Antileukemic Activities

Abstract

Escherichia coli-derived l-asparaginase (EcA) is a crucial frontline therapy for the treatment of acute lymphoblastic leukemia (ALL), yet its use in clinical settings remains considerably compromised due to its immunogenicity, hypersensitivity reactions, hepatotoxicity, rapid proteolytic degradation, and a markedly short plasma half-life. However, given the clinical benefits of l-asparaginase, an improved variant of it, combined with a biocompatible immobilizing agent, can plausibly circumvent the drawbacks mentioned above. Herein, this study, in order to mitigate these limitations, first, we rationally developed a less immunogenic variant of l-asparaginase, namely, KHY-17-EcA, by a site-directed mutagenesis approach, and then we utilized biocompatible carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs) to covalently immobilize the KHY-17-EcA variant along with the WT-EcA. The resulting nanobiocatalysts (MWCNT-WT-EcA and MWCNT-KHY-17-EcA) exhibited a 96% enzyme-immobilizing capacity. Interestingly, these two immobilized enzymes exhibited excellent thermal and serum stability as well as superior catalytic activity across a wide range of pH and temperature conditions compared to their free forms. Notably, both immobilized enzymes retained almost 90% of their original activity after 4 weeks of storage and exhibited complete resistance to proteolytic degradation by asparaginyl endopeptidase and cathepsin B after 24 h of incubation. Furthermore, the immobilized KHY-17-EcA variant exhibited significantly reduced glutaminase activity, a low Km of 0.312 mM, and high substrate specificity, along with the potent cytotoxic activity against human leukemia cell lines (Jurkat, MOLT-4, K562), while sparing noncancerous human bronchial epithelial cells (HBEC-5i). Apoptosis analysis of treated leukemia cells revealed marked nuclear morphological alterations and a significant increase in the level of apoptotic cell death. These findings collectively demonstrate that the newly synthesized MWCNT-KHY-17-EcA nanobiocatalyst has the potential to be utilized in leukemia treatment. Taken altogether, these results highlight the efficacy of MWCNT-based immobilization as a robust strategy to prolong enzyme shelf life, confer resistance to proteolytic degradation, and potentiate biological functionality, thereby broadening its translational scope in both biotechnology and cancer therapeutics.