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Linker Engineering and SA?PVA Immobilization for High?Efficiency Carcinine Synthesis. The solubility of Ebony?sfp was enhanced by linker peptide L2 in WSL2E, and the WSL2E was immobilized by SA?PVA to yield WSL2E@SA?PVA, achieving 71.13?mM carcinine in fed?batch catalysis.ABSTRACTCarcinine, a valuable imidazole dipeptide with antioxidant and therapeutic properties, faces biosynthesis challenges due to enzyme aggregation and substrate inhibition. In this study, an integrated strategy combining linker peptide engineering and immobilization was applied to address these challenges and enhance carcinine production. Rational design of linker peptides (D5, L2, L3) in the sfp?Ebony fusion protein enabled its highest soluble expression in WSL2E strain, achieving 93.1% conversion efficiency—3.5?fold higher catalytic efficiency than WSGE strain. Response surface methodology optimized sodium alginate?polyvinyl alcohol (SA?PVA) immobilization parameters (5% PVA, 3% SA, 2.3% CaCl?), yielding excellent immobilized WSL2E@SA?PVA cells with 95.93% activity recovery. Structural characterization by scanning electron microscopy (SEM), Fourier?transform infrared spectroscopy (FT?IR), and X?ray diffraction (XRD) confirmed the formation of a porous SA?PVA matrix that protected cells from harsh conditions. The immobilized biocatalyst exhibited superior operational stability (retaining >?80% activity after 7 cycles) and storage stability (maintaining 44.89% activity after 14 days at 4°C). Fed?batch scale?up (50?mL) achieved a record carcinine titer of 71.13?mM, mitigating the inhibitory effect of high substrate concentrations through phased substrate feeding. This study provides a scalable biocatalytic platform for industrial carcinine production, effectively addressing key bottlenecks in biocatalyst stability and substrate tolerance.