Nanoparticle-supported consecutive reactions catalyzed by alkyl hydroperoxide reductase

Multi-enzyme systems have been widely employed in biotransformations to produce a variety of useful compounds. An efficient and stable multi-enzyme system is often required for large-scale applications. Herein we report the immobilization of a multi-enzyme system, which catalyzes consecutive reactions by alkyl hydroperoxides reductase (AhpR) on functionalized single-walled carbon nanotubes (SWCNTs). AhpR, composed of H2O2-forming NADH oxidase (nox) and peroxidase (AhpC), protects microorganisms from the toxic effects caused by organic hydroperoxides and regulates H2O2-mediated signal transduction. Both His-tagged nox and AhpC were immobilized via non-covalent specific interactions between His-tagged proteins and modified SWCNTs. The activity and stability of AhpR at different nox/AhpC ratios were examined and the immobilized AhpR system demonstrated ca. 87% of the native enzyme activity. We found that various nox/AhpC ratios may affect overall AhpR activity but not the total turnover number. The amount of intermediate hydrogen peroxide is not influenced by immobilization and it decreases when the weight of AhpC increases, and becomes undetectable when nox/AhpC ratio reaches above 1:50. Hence, we believe that this non-covalent specific immobilization procedure can be applied to multi-enzyme systems with satisfactory activity retention and stability improvement during consecutive reactions.