Integrated Asparaginase Development for Biomedicine and Food Safety: from Protein Engineering to AlphaFold-guided Oligomerization Prediction

Asparaginase (ASNase), an enzyme that hydrolyzes l-asparagine into l-aspartic acid and ammonia, represents an essential biocatalyst at the interface of biomedicine and food safety. Clinically, ASNase is a cornerstone therapy for acute lymphoblastic leukemia (ALL), while in the food industry it is widely applied to reduce acrylamide formation, thereby lowering neurotoxic and carcinogenic risks without compromising product quality. However, despite separate advancements in using ASNase for cancer therapy and food safety, an integrated optimization strategy is still lacking. Since both applications rely on the same catalytic mechanism, require good biochemical properties (e.g., Km, Kcat, Vmax, and half-life), and share critical production safety concerns, a dual-purpose enzyme could be engineered for both applications. This review bridges this development gap by proposing an integrated framework that unifies structure–function analysis and protein engineering strategies for both applications, enabling cost- and time-efficient workflows starting with bioinformatic evaluation. Ultimately, this review outlines strategies to engineer ASNase variants with enhanced catalytic efficiency, stability, and reusability, alongside reduced glutaminase co-activity and immunogenicity. This review evaluates emerging approaches, including humanized design, rational engineering, AlphaFold 3-based structural and dimerization predictions, and encapsulation, alongside advanced fermentation platforms for cost-effective and large-scale production. By assessing the correlations between enzymatic potency, structure–function relationships, immunogenicity, and industrial viability, this multidisciplinary work integrates bioengineering, computational modeling, and fermentation development to guide future ASNase innovations in healthcare and the food industry. Integrates medical and food ASNase frameworks for cost- and time-effective development. Compiles recent mutational data guiding the rational design of optimized, multi-purpose ASNase variants. Demonstrates the ability of AlphaFold 3 to predict ASNase structural heterogeneity and oligomerization states. Assesses innovative fermentation, humanization, immobilization, and encapsulation of ASNase.