Diatoms are single‐celled microalgae that produce a large variety of hierarchically porous, silica‐based microparticles as cell wall material. The presence of genetically encoded silica nanopatterns endows the biosilica with favorable properties for a wide range of applications including catalysis, chemical sensing, photonics, and drug delivery. Enhancing the performance of diatom biosilica requires i) a better understanding of the structure–property relationship in this material, and ii) methods that enable the manipulation of the biosilica structure and properties in a targeted manner. Here, genetic engineering of the diatom Thalassiosira pseudonana is employed to immobilize enzymes (glucose oxidase and horseradish peroxidase) into structurally distinct regions of the biosilica, which are termed valves and girdle bands. Remarkably, glucose oxidase in girdle bands exhibits >3‐fold higher catalytic activities compared to its location in valves. It is demonstrated through enzyme accessibility studies, protein engineering, and genetic engineering of biosilica morphology that the divergent enzyme activities are caused by the differences in the inherent silica nanopatterns of valves and girdle bands. This work highlights the importance of silica nanoscale architecture for the activity of immobilized enzymes and provides unprecedented tools for the biotechnological production of silica microparticles with tailored catalytic activities and anisotropic functionalities.
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