Live Diatom Silica Immobilization of Multimeric and Redox-Active Enzymes

Abstract

ABSTRACTLiving organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biomineralsin vitroand endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materialsin vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO2(silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatomThalassiosira pseudonanawas accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. Thein vivoincorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct forin vivoimmobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.