Engineering affinity tag strategies for selective downstream processing of H₂O₂-generating oxidases: Minimizing...

Hydrogen peroxide (H₂O₂) is a key green oxidant for industrial biotransformations, yet its conventional production, storage, and handling present significant safety, logistical, and sustainability challenges. Enzymatic in situ generation using oxidases has emerged as an attractive alternative, enabling controlled H₂O₂ supply, eliminating transport requirements, and improved process integration. However, co-purified catalase from microbial expression hosts remains a major bottleneck, as it rapidly decomposes H₂O₂ and severely compromises process efficiency. This work demonstrates an affinity-driven downstream processing strategy to selectively minimize catalase contamination and enhance the performance of H₂O₂-generating biocatalysts. The bacterial oxidase N-acetylglucosamine oxidase (NagOX) was used as a model enzyme and genetically fused to different affinity tags, including His₆-tag and carbohydrate-binding modules (CBM3 and CBM9). Their impacts on enzyme production, purification, stability, and immobilization were systematically evaluated from a bioprocess engineering perspective. While conventional His₆-tag-based IMAC purification resulted in substantial residual catalase activity, the CBM3 affinity platform enabled selective adsorption onto inexpensive cellulose supports, achieving minimized contamination, with no detectable catalase activity under the assay conditions used in a single step. This approach provided a threefold higher purification factor (11.7 vs 4.0) and increased apparent oxidative activity by 42%, by eliminating competing side reactions. In contrast, CBM9 fusion led to structural instability and proteolytic degradation. Importantly, the CBM3-based strategy integrates purification, immobilization, and catalyst recovery into a single, scalable downstream operation using low-cost materials. The resulting biocatalyst exhibited enhanced performance for in situ H₂O₂ generation, highlighting the potential of affinity tag engineering for efficient and industrially relevant oxidase-based bioprocesses.