B CUBE hosts the 4th Scientific Advisory Board meeting
20 years biotechnology initiative in Saxony
Kröger Group - Bionanotechnology
A remarkable characteristic of biological formation of inorganic materials (biomineralization) is the precise control of complex mineral structures in three dimensions and over several orders of magnitude in length scale (from nanometers up to millimeters and beyond). This is achieved by the action of highly organized assemblies of cellular macromolecules within specific intracellular organelles, on the cell surface, or within specialized tissues, which enable mineral formation to proceed under mild (i.e., physiological) reaction conditions. The Kröger group aims to utilize insight from their research on the molecular mechanism of diatom silica biomineralization (see Biomineralization) to implement novel routes for syntheses of functional inorganic materials with controlled nanoscale architectures.
Based on the structure of silaffins, a group of silica forming proteins from diatoms, the Kröger group has developed recombinant proteins that can be applied for the synthesis of silica and titania materials from aqueous solution at near neutral pH and room temperature (collaboration with Ken Sandhage, Georgia Tech, Atlanta USA).1 Remarkably, the recombinant silaffin rSilC is able to induce the formation of unique rutile TiO2 structures from an aqueous solution of the Ti(IV) complex Ti(IV)-bis-lactato dihydroxide (TiBALDH; Figure 1).
Figure 1. Structure of rSilC-induced titania. (A) The recombinant silaffin polypeptide, rSilC, induces the formation of spherical titania particles (SEM image, right) form an aqueous solution of TiBALDH (chemical structure, left) at pH 7. (B) Close-up TEM image, (C) selected area electron diffraction image, (D) TEM lattice fringe image. (E) Powder X-ray diffraction spectrum.
The work on recombinant silaffins has revealed that peptides carrying a sufficiently high positive charge density are generally capable of silica and titania formation for silicic acid or TiBALDH solutions, respectively.2 As a low-cost alternative for recombinant silaffins, the commercially available peptide protamine (a highly abundant DNA binding protein from fish) used to develop a layer-by-layer (LbL) mineralization technique (collaboration with Ken Sandhage, Georgia Tech, Atlanta USA) (Figure 2A). This method enabled the deposition on negatively charged surfaces (e.g., silicon wafers, silica spheres, diatom silica) of continuous and conformal nanoscale mineral layers in 5 nm (for silica) and 7 nm (for titania) increments per cycle (Figure 2B).2
Figure 2. LbL Mineralization. (A) Schematic of the process. The green threads represent protamine molecules. (B) Thickness of silica (black) and titania (grey) bearing layers deposited on silicon wafers via protamine-mediated LbL mineralization (analysis by atomic force microscopy).
The mild conditions for mineral deposition allowed for the incorporation of active enzymes into the mineral layers in situ. To achieve efficient incorporation, the enzyme needs to be linked to a silica-binding tag, which was achieved via covalent cross-linking of the enzyme with protamine. The immobilized enzyme was active and substantially stabilized against thermal denaturation and proteolytic attack compared to the free enzyme in solution.3
Figure 3. Enzyme immobilization through LbL mineralization. The enzyme glucose (GOx) oxidase was cross-linked to protamine (PA) yielding GOx-PA, which exhibits the same specific activity as GOx.3 GOx-PA binds to silica and titania surfaces at pH 7.0 and retains ~75 % of its activity after it has been encased by a nanoscale layer of silica or titania. 3
The silica structures produced by diatoms are low-cost materials that exhibit many features interesting for a broad range of nanotechnological applications including membranes for biomolecule separations, size-selective sensors, microelectro-mechanical systems (MEMS), and masks for microfabrication. To fully exploit the nanotechnological potential of diatom silica, the Kröger group has recently developed a novel method to introduce additional functionalities into the diatom silica. This functionalization method is based on molecular genetic engineering of the silica biomineralization process in the diatom T. pseudonana. Recombinant genes that encode silaffin Sil3 fused to a functional protein of choice, have been incorporated into the T. pseudonana genome through techniques established by the Kröger group. The silaffin-domain is responsible for targeting the fusion protein for incorporation into the forming silica, thus stably immobilizing the functional domain in the frustule after completion of silica biogenesis (Figure 4). The method has been coined Live Diatom Silica Immobilization (LiDSI) , and has been successfully used to immobilize in the diatom silica various enzymes including cofactor-dependent and multimeric enzymes.4,5 Attachment to the biosilica had a stabilizing effect on the enzymes.4,5
Figure 4. Live diatom silica immobilization (LiDSI) of enzymes. The method has shown to be applicable to the following enzymes: hydroxylaminobenzene mutase B (HabB)4, b-glucuronidase5, glucose oxidase5, galactose oxidase5, horseradish peroxidase5.
The method represents a new paradigm for the sustainable bio-enabled production of complex nanostructured materials with tailored functionalities. A compelling advantage of LiDSI is that the enzyme does not need to be isolated prior to immobilization. This should be particularly advantageous for enzymes that are expensive to isolate and prone to rapid denaturation in vitro. Because of the excellent mechanical and mass transport properties of diatom silica enzyme-functionalized diatom cell walls may be attractive materials for flow-through devices from very small (microfluidics) to large scale (flow-through reactors).
- N. Kröger, M. B. Dickerson, G. Ahmad, Y. Cai, M. S. Haluska, K. H. Sandhage, N. Poulsen, V. C. Sheppard (2006) Angew. Chem. Int. Ed. 45, 7239-43.
- Y. Fang, Q. Wu, M. B. Dickerson, Y. Cai, S. Shian, J. Berrigan, N. Poulsen, N. Kröger, K. H. Sandhage (2009) Chem. Mater. 21, 5704-10.
- N. R. Haase, S. Shian, K. H. Sandhage, N. Kröger (2011) Adv. Funct. Mater. 21, 4243-51.
- N. Poulsen, C. Berne, J. Spain, N. Kröger, (2007) Angew. Chem. Int. Ed. 46, 1843-46.
- V. C. Sheppard, A. Scheffel, N. Poulsen, N. Kröger (2012) Appl. Environ. Microbiol. 78, 21-8.