Development of a pyridoxine 4-oxidase nanoreactor for oxidative therapy

Oxidative therapy for cancer treatment represents a promising application of nanoreactors in medicine, but there is still a lack of systems to deliver the oxidative enzymes to target tissues. Encapsulins, a novel class of nanocompartments found in bacteria and archaea, can encapsulate specific enzymes, offering significant potential for developing nanoreactors. This study explored the development of a nanoreactor based on the bacterial enzyme pyridoxine 4-oxidase (PNOX). PNOX catalyzes the oxidation of pyridoxine to pyridoxal forming H₂O₂. PNOX was loaded into the encapsulin from Myxococcus xanthus, whose pore diameter was enlarged by engineering the loop that forms the rim of the pore. Wild-type and engineered encapsulins were loaded with PNOX, and it was found that two variants of the poreengineered encapsulins showed an increase in specific activity from 1.5 μM min^-1 mg^-1 in the wild-type encapsulin to 4.8 and 10.6 μM min^-1 mg^-1. The encapsulated cargo exhibited remarkable stability, retaining over 90 % of its enzymatic activity after 4 h at 37 °C with half-lives of 20 h. These results demonstrated that engineering the pore structure of encapsulins enhances the mass transfer efficiency of substrates and products, and gives new insights into the design and applications of nanoreactors.