Reactive　oxygen　species　(ROS)　are　major　contributors　to　radiation　therapy-induced　side　effects　in　cancer　patients.　A　fusion　antioxidant　enzyme　comprising　glutathione　S-transferase　(GST),　superoxide　dismutase　1　(SOD1),　and　a　transmembrane　peptide　has　been　shown　to　effectively　mitigate　ROS-induced　damage.　To　enhance　its　targeting　capability,　the　fusion　protein　was　further　modified　by　incorporating　a　matrix　metalloproteinase-2/9　substrate　peptide　(X)　and　the　transmembrane　peptide　R9,　yielding　the　antioxidant　enzyme　GST-SOD1-X-R9　(GS1XR).　This　modification　reduced　its　transmembrane　ability　in　tumor　cells,　thereby　selectively　protecting　normal　cells　from　oxidative　stress.　However,　the　use　of　non-human　GST　poses　potential　immunogenicity　risks.　In　this　study,　we　employed　seamless　cloning　technology　to　construct　an　expression　vector　containing　the　human　GST　gene　to　replace　the　non-human　GST　gene,　and　then　expressed　and　purified　novel　fusion　antioxidant　enzymes　GS1R　and　GS1XR.　The　protective　effects　of　newly　constructed　GS1R　and　GS1XR　against　hydrogen　peroxide　(H2O2)-induced　oxidative　damage　in　L-02　cells　were　then　evaluated　using　GS1　as　a　control.　Enzymatic　activity　assays　revealed　that　the　specific　activity　of　GST　in　GS1XR　remained　unchanged　compared　to　the　unmodified　protein,　while　SOD　activity　was　enhanced.　Exposure　to　200　μmol/L　H2O2　transiently　activated　the　nuclear　factor-erythroid　2-related　factor　2　(Nrf2)　pathway;　however,　this　activation　diminished　after　24　h,　reducing　cell　viability　to　48.4%.　Both　GS1R　and　GS1XR　effectively　scavenged　intracellular　ROS,　directly　counteracting　oxidative　stress　and　promoting　Nrf2　nuclear　translocation,　thereby　activating　antioxidant　pathways　and　restoring　cell　viability　to　normal　levels.　The　two　enzymes　showed　comparable　efficacy.　In　contrast,　GS1,　lacking　transmembrane　capability,　was　restricted　to　scavenging　extracellular　ROS　and　provided　only　limited　protection.　In　conclusion,　both　novel　fusion　antioxidant　enzymes　demonstrated　significant　potential　in　safeguarding　normal　cells　from　ROS-mediated　oxidative　damage.　The　findings　provide　a　foundation　for　further　investigation　in　related　field.　©2025　Chin　J　Biotech,　All　rights　reserved.