Enhanced Catalytic Performance of P450 Monooxygenase CYP11B1 by Constructing Fusion Protein with Preferred Reductase...

As promising biocatalysts, cytochrome P450 enzymes (CYP450s) participate in various reactions involving complex organic compounds. However, their broader application potential is constrained by the dependence on reductase domains. In this study, we engineered a self-sufficient P450 chimeric enzyme by fusing 11β-hydroxylase CYP11B1 with distinct reductase domains from P450_BM3, P450_RhF, and CYP116B3 through peptide linkers. The results demonstrated that the catalytic efficiency of the chimeric CYP11B1-BMR exceeded that of "natural" electron transfer systems CYP11B1-AdR/Adx by approximately 25%. Under optimized expression conditions, the activity of CYP11B1-BMR using 7-ethoxycoumarin and cytochrome c as substrates increased by 30% and 25%, respectively. Among the various linkers tested for rigidity and length, a flexible linker comprising 15 amino acids yielded the highest reduction activity for CYP11B1-BMR, which was 26.6% greater than that observed for CYP11B1-AdR/Adx. This study not only successfully developed a self-sufficient chimeric enzyme but also enhanced its catalytic performance, thereby providing a practical approach for constructing more efficient biocatalytic systems.