An effective enzymatic assay for pH selectively measuring direct and total bilirubin concentration by using of CotA

In this study, we aimed to develop B. subtilis spore coat protein A (CotA) for the enzymatic determination of bilirubin. Firstly, molecular docking and oxidation kinetic analysis confirmed the feasibility of CotA for oxidizing bilirubin. Secondly, CotA showed pH-preferable oxidization performance to direct bilirubin (DB) in acidic conditions and an alkaline-catalytic oxidation capacity to total bilirubin (TB). Mechanism analysis results confirm that the conformational changes of CotA, DB and UB caused by pH changes are responsible for the selective oxidation of DB and TB by CotA. Then, CotA exhibits better structural characteristics and enzymatic performance than M. verrucaria-derived bilirubin oxidase (Mv-BOD). Besides, the strong anti-interference ability helps CotA adapt to complex catalytic environment in the detection of DB and TB. Our results prove that CotA can be used as a promising candidate bio-enzymatic detection reagent for DB and TB.