Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119

Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon–hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H 2 O 2 ) as an oxygen and electron donor, but the reaction with H 2 O 2 is inefficient. The development of P450s that can use H 2 O 2 will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H 2 O 2 -dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius , with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher k cat for Amplex Red peroxidation and twofold higher k cat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H 2 O 2 . While the K m for H 2 O 2 and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity. Graphic abstract