Nanoconfined proteins and enzymes

Once the proteins are confmed within the porous structure of the sol-gel matrix, it becomes imperative to determine if these nanostructured materials still remain amenable to interactions with external reagents. This issue assumes central importance because the functional relevance of nanoconfined proteins and enzymes is contingent upon their reactions with suitable substrates or ligands. 

Nanostructured materials obtained by sol-gel encapsulation of biomolecules are a novel class of biomaterials. The biological macromolecules, confined within the nanometer-size pores of the matrix, show both similarities to and differences from solution characteristics. The effects of nanoconfinement on the structural and reactivity patterns of the proteins and enzymes are discussed. The applications of these nanostructured biomaterials in the area of molecular biorecognition, detection, and biosensing are also presented. 

The paper is organized in three parts. First, the effects of nanoconfinement on the structure of the sol-gel trapped biomolecule are discussed. Second, from the results on apparent reactivity of these biomaterials, the effects of the matrix on the reactivity of trapped enzyme are elucidated. Finally, the interaction of the confined biomolecules with exogenous ligands/substrates and the applications of these materials in the area of molecular biorecognition are discussed. The reaction chemistries of biologically active molecules in die nanostructured materials have been crucial in establishing the role of the matrix upon the structure and reactivity of the confined proteins. 

Oxalate Oxidase. For the oxalate oxidase system, the value of the Michaelis constant (Km) increased for the sol-gel nanoconfined system (Km /lO = 1.1 in solution and 4.1 in aged gel). The approximately fourfold increase indicates that the binding of the oxalate with the enzyme is weaker. The apparent association constant (kcat/ m) for the oxalate-oxalate oxidase complex was substantially altered as a result of confinement of the protein the sol-gel matrix (kcat/Km = 170 in solution and 2.3 in aged gel). This implicates a relatively destabilized enzyme-substrate complex. Additionally, the kcat parameter is also reduced in the gel (kcat /lO" = 187 in solution and 9.4 in aged gel), suggesting deactivation of the product forming step in the gel medium. In this case, whole the dissociative product forming step is reduced only by a factor of 20 (187/9.4), the associative step is reduced considerably by a factor of 75 (170/2.3). 

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