Superoxide dismutase from Citrus limon (SOD_Cl) is an antioxidant enzyme that remains stable in acidic environments and under proteolytic degradation due to the stability of its loop IV structure. However, its oral bioavailability is limited because of its molecular size. To improve intestinal permeability, SOD_Cl was fused with a gliadin-derived peptide (QQPYPQPQPF), resulting in the fusion protein GliSOD_P61. This study aimed to evaluate the effects of peptide fusion on protein activity, stability, and the impact of copper (Cu) ion supplementation on enzyme activity. Recombinan protein were expressed in E. coli BL21(DE3) with Cu and Zn ions added to the growth medium. Proteins were purified using Ni-NTA affinity chromatography, and enzymatic activity was assessed using the NBT-riboflavin method. Cu and Zn content were quantified via ICP-OES. Results showed that Cu ion supplementation in the medium reduced protein expression by approximately 40-50%. SOD_Cl exhibited higher stability and activity compared to GliSOD_P61 under acidic and proteolytic conditions. Fusion with the gliadin peptide decreased specific activity and stability. Cu levels in SOD_Cl remain unchanged with Cu addition, while GliSOD_P61 showed increased Cu content and enzymatic activity in vitro. In conclusion, peptide fusion reduced SOD_Cl activity and stability, but in vitro addition significantly enhanced GliSOD_P61 activity.