| Summary: | The development of solid biocatalysts has rapidly progressed for applications in nanomaterials. The immobilization of enzymes in solid materials is considered an alternative method for constructing traditional biocatalysts with high stability and efficiency to be reused. However, the design of solid scaffolds from nanoporous materials to immobilize multiple enzymes and improve catalytic reactivity remains challenging. In this study, the engineering of in-cell protein crystals is demonstrated to have the potential for immobilizing two enzymes that promote a cascade reaction. Polyhedra is known as an in-cell crystalline protein produced in insect cells by the infection of cytoplasmic polyhedrosis virus. We constructed the 38-residue-deletion mutant, which forms interlinked hollow nanocages with a diameter of 5 nm in the crystal. The mutant crystal can encapsulate Candida antarctica lipase B and Lactobacillus kefir alcohol dehydrogenase. The composite crystal significantly enhances the reactivity of the cascade reaction with 1.9-fold and 3.8-fold higher efficiency than that of the wild-type crystal and the mixture of the free enzymes, respectively. The higher reactivity is because of the substrate and intermediate efficiently diffusing through the extended channels designed within the nanoporous crystal. These results suggest the possibility of using polyhedrin crystals to design the nanoporous materials for developing further applications in bio-nanomaterial science.
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