The role of linker on the elevated biosensing performance of a carbohydrate-binding module-tagged sarcosine oxidase.

Creatinine is a pivotal biomarker for renal function assessment, and developing its rapid, sensitive, stable point-of-care testing (POCT) biosensors remains critical. Enzyme-based biosensors using sarcosine oxidase (SOX) in a three-enzyme cascade are promising, but challenges persist in post-immobilization enzymatic activity and long-term stability, especially for low-activity SOX. Herein, we report rational engineering of carbohydrate-binding module 3 (CBM3)-fused SOX variants to address these limitations. CBM3 was genetically fused to N-/C-termini of monomeric SOX via distinct linkers (proline-rich XP for N-terminal, α-helical EAAK for C-terminal), generating CBM3-XP-SOX and SOX-EAAK-CBM3. Comparative analysis showed SOX-EAAK-CBM3 exhibited superior soluble expression (15.96 mg/mL), enhanced ambient-temperature catalytic activity (39.96%-56.23% higher than WT SOX), and exceptional electrochemical stability (25 days, R2 > 0.99). In contrast, CBM3-XP-SOX suffered linker cleavage and structural instability, limiting stability to 2 days. Structure-guided linker engineering of the N-terminal fusion identified unstable K-E-P motif in XP linker; truncation and flexible glycine introduction yielded CBM3-XG-SOX. The CBM3-XG-SOX/cellulose/Pt biosensor showed 7-fold improved electrochemical stability (15 days) vs. parental CBM3-XP-SOX, with rapid response (19-30 s) and strong anti-interference against physiological interferents. Serum and urine real-sample analysis confirmed high accuracy (recovery: 96.28%-97.88%) and superior precision vs. commercial kits, with narrower errors for low or high creatinine concentrations. This work establishes a rational framework for fusion enzyme engineering via bioinformatics-guided linker/fusion site optimization, overcoming low-activity enzyme-based biosensor limitations. CBM3-fused SOX biosensors enable cost-effective, stable, sensitive POCT creatinine detection, with broad clinical diagnosis and home health monitoring applications.