Epoxy-norbornane (EPO-NBE) is a crucial building block for the synthesis of various biologically active heterocyclic systems. To develop an efficient protocol for producing EPO-NBE using norbornene (NBE) as a substrate, cytochrome P450 enzyme from Pseudomonas putida (CYP238A1) was examined and its crystal structure (PDB code: 7X53) was resolved. Molecular mechanism analysis showed a high energy barrier related to iron-alkoxy radical complex formation. Therefore, a protein engineering strategy was developed and an optimal CY-P238A1 NPV variant containing a local hydrophobic "fence" at the active site was obtained, which increased the H 2 O 2 -dependent epoxidation activity by 7.5-fold compared with that of CYP238A1 WT . Among the "fence", Glu255 participates in an efficient proton transfer system. Whole-cell transformation using CYP238A1 NPV achieved an EPO-NBE yield of 77.6 g • L À 1 in a 30-L reactor with 66.3 % conversion. These results demonstrate the potential of this system for industrial production of EPO-NBE and provides a new biocatalytic platform for epoxidation chemistry.