Cellulose-Binding Module-Mediated Immobilization of l-Arabinose Isomerase for Reusable d-Tagatose Production

The development of stable and recyclable biocatalysts is vital for sustainable bioprocessing. In this study, we demonstrated fusion of l-arabinose isomerase from Thermotoga neapolitana (TnAI) with a cellulose-binding module (CBM) to facilitate direct, noncovalent immobilization on cellulose fibers for the biocatalytic transformation of d-galactose to d-tagatose. The impact of His-tag positioning on TnAI activity and quaternary structure was initially assessed, revealing that the C-terminal tag caused enzyme aggregation and loss of activity. In contrast, the N-terminal His-tag preserved the hexameric configuration and catalytic efficiency. Three CBM-TnAI fusion variants containing flexible linkers (L1-L3) were subsequently generated, expressed, and purified. All CBM-fused variants retained solubility and catalytic activity comparable to the native enzyme, confirming that the N-terminal CBM fusion does not disrupt active-site conformation. Immobilization studies on microcrystalline cellulose showed high binding efficiencies (>80%) and substantial retention of catalytic activity, with CBM_L2_TnAI exhibiting the highest cellulose-binding efficiency (>90%) and immobilized activity (∼87%). Consistent with its superior catalytic performance and immobilization characteristics, CBM_L2_TnAI was selected for reusability studies and retained approximately 40% of its initial activity after five reaction cycles, demonstrating good operational stability. This CBM-mediated immobilization strategy eliminates the need for chemical cross-linkers, providing a simple, cost-effective, and sustainable platform for producing reusable enzyme systems. The cellulose-protein hybrid catalyst developed herein offers a versatile framework for continuous or batch biotransformation of low-value waste carbohydrates into value-added rare sugars.