Construction of P450 scaffold biocatalysts for the biodegradation of five chloroanilines.

Chloroanilines represent a class of persistent and highly toxic environmental pollutants, posing significant challenges for green remediation strategies. While P450BM3 monooxygenases are renowned for their ability to catalyze the monooxidation of inert C-H bonds, costly NAD(P)H and complex electron transport systems required for P450BM3 catalysis limit their practical applications. This study pioneers the development of innovative artificial biocatalysts by strategically engineering the active site of P450BM3. Specifically, the substitution of the highly conserved threonine 268 with aspartic acid effectively induces peroxygenase activity, allowing for enhanced catalytic efficiency. Remarkably, the engineered P450BM3 mutants achieved degradation rates of 98.38-99.18 % for five chloroanilines (4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, and 3,5-dichloroaniline) in just 10-15 min, all without the need for NAD(P)H or dual-functional small molecules. Comprehensive degradation mechanism analysis via UPLC-MS corroborated the remarkable performance of these biocatalysts. This research not only demonstrates a novel approach for engineering P450 monooxygenases to exhibit peroxygenase activity but also significantly broadens their potential applications in synthetic chemistry and synthetic biology, paving the way for greener and more sustainable remediation technologies.