Structural-guided high stable fusion urate oxidase engineering to self-assemble with cellulose modified electrode for...

The high stability and preserved catalytic activity of immobilized urate oxidase (UOX) are key toward the precise sensing of uric acid (UA). Here an electrochemical UA biosensor based on self-assembly immobilization of UOX on a cellulose-modified electrode upon fusing a carbohydrate binding module (CBM) to facilitate the detection of UA. With the aid of a computational structural-guided enzyme engineering strategy, the structural stability of N-terminus CBM3 fusion was proved to be superior to CBM3 or CBM2 fused to the C- terminus of UOX. The residues in the N-terminus of CBM3 (P2VSG5) and in the linker region (K158EPMSN163) predicted harmful for the structural stability of CBM3-UOX were furtherly deleted (CBM3-Δ10aa-UOX), which greatly promoted the reliability of the UA biosensor with linearity in the range of 0 to 0.625 mM UA with an R² > 0.99 for up to 20 d compared with glutaraldehyde cross-linked UOX biosensor (0-0.375 mM, 5 d). In addition, the mechanisms of the increased current observed during the initial period for fusion UOXs were explored and attributed to the interaction between the UOX subunits and linker breakage as verified by co-immobilization of wild-type UOX and CBM3-Δ10aa-UOX (0-0.875 mM, > 34 d, R² > 0.99), furtherly proving the enhanced stability of the link-peptide of CBM3-Δ10aa-UOX to prolong the current increase process and improve the working life. This study emphasized the effectiveness of self-assembly immobilization toward advancing the biosensing field and provided a robust strategy for the evolution of similar multimeric biosensors.