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4 Repeat Proteins as Scaffolds for Biomolecular Patterning

4 Repeat Proteins as Scaffolds for Biomolecular Patterning

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4



Designed Repeat Proteins as Building Blocks for Nanofabrication



71



Fig. 4.8 Schematic representation of bottom-up

approach for the fabrication of functional supramolecular architectures. (a) Schematic representation of

different elements with great potential for applications in

different nanotechnological fields, such as nanoparticles,

metal ions, small organic molecules, and functional peptides or proteins. (b) Schematic representation of different

scaffolding units such as small organic templates, DNA,

peptides and proteins. In this chapter we will focus on

proteins as scaffolds that can be modified with different



reactivities. The colored surfaces represent orthogonal

reactivities that will be used to conjugate the different

components. (c) Conjugates in which different components are linked to the scaffolding units by the selected

reactivity. (d) Supramolecular hybrid architectures formed

by the arrangement different functionalized scaffolds. The

scaffolding units are used to arrange active components

into defined patterns, as required to achieve optimal properties in the final structure



un-natural amino acids are incorporated into proteins, expanding the potential reactivities of proteins for bioconjugation. These strategies allow

the introduction of specific binding properties to

conjugate any metallic nanoparticle or even

explore organo-metallic strategies. Moreover, the

recent developments of in situ reduction of metallic salts into nanoclusters or nanoparticles in the

presence of protein open the field of a one-pot

fabrication of nanowires by a combination of

metal salts, scaffolding proteins and reducing

conditions, followed by thermal annealing of

metal growth process.

Hybrid materials based on chiral molecules

and metallic nanostructures are also of interest in

chiral plasmonics applications [67], non-linear

optics [68], or negative refractive index matter

[69]. One example is the use of supramolecular

patterning to create novel chiral superstructures

of gold nanorods [70]. Also the preparation of

metamaterials based on plasmonic mesophases

with switchable polarization-sensitive plasmon

resonances shows several potential applications

strate [173] or

in combination with adhesion-promoting layers

such as secondary cell wall polymers [66, 67, 138,

150], lipids [31, 151], polyelectrolytes [151] or

complete ultrafiltration membranes [203]. These

sensory-active materials can also be comprised of

commercial synthetic polymers and S-layer proteins, which in turn can be further chemically

modified. To ensure their functionality, the introduction of additional spacer molecules has proven

to work in some cases [66, 67, 96]. In many

instances, these modifications can have a positive

effect on both the stability and sensitivity of the

sensory-active layers. In turn, interactions

between the sensor and the respective target molecules are capable of generating signals that can

be converted on a sensor chip with a suitable

transducer in measurable, mostly electrical signals, which can be read out after appropriate

amplification. These optical, electrical or mechanical signals can be determined via optoelectronic,

amperometric, potentiometric or piezoelectric



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S-Layer-Based Nanocomposites for Industrial Applications



methods [203]. Extensive work on the detecting

principles of sensory-active S-layer proteins

based constructs—especially with respect to medical diagnostic applications—is highly illustrative

(e.g. [180, 201, 203]). For example, recent publications show the potential of sensor chips in

determining various sugars [136, 203].

In addition to planar layer arrangements,

S-layer proteins are also suitable for covering 3D

sensory structures such as gold or silver nanoparticles [100, 101], glass fibers [173] or carbon

nanotubes (CNT) [133]. Various metals or metalloids (e.g. arsenic) have been detected in an aqueous medium using S-layer protein- functionalized

gold nanoparticles of different sizes with a simple colorimetric assay (Fig. 11.4) [100]. The

potential of developing enhanced biosensors with

increased sensitivity and selectivity, as well as

lower production and maintenance costs, is

dependent on advances in S-layer proteins. In

fact, next-generation biosensors are likely to be

miniaturized, highly integrated, able to measure

in real time with little oversight, and will either

be regenerative or available as cost-effective disposable modules.

Fig. 11.4 Detection of

solubilized arsenic (V)

using S-layer

functionalized AuNP

(50 nm in diameter). (a)

colorimetric assay, (b)

UV/VIS measurements

[100]



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11.3.4 S-Layer Proteins as Matrix

in Bio-Mineralization

and Production

of Nanoparticles

Using the regular charge clusters on their surface,

S-layer proteins are capable of binding different

ions, which can be converted to inorganic nanostructures and nanoparticles under appropriate

conditions [201]. The natural protective function

of these microorganisms against toxic environmental influences can be exploited for the synthesis of nanoparticles and nanoparticle arrays or to

separate prefabricated particles. Nanoparticles

often show altered electrical, optical or chemical

properties—in contrast to what has been observed

in bulk materials. One problem that often occurs

is the tendency for such a colloidal system to

aggregate into larger particles. This disadvantage

can be overcome by using S-layer proteins as a

suitable substrate for a variety of well defined,

highly dense, and tightly immobilized in situ or

pre-synthesized nanoparticles. Due to their

intrinsically low surface-to-volume ratio, such

immobilized nanoparticles are ideal candidates



254



for catalytic and sensor applications. In fact, the

first promising results were obtained with Pt

nanoparticles for the oxidation of carbon monoxide [68]. Recently, [174] described the use of

photocatalytically active TiO2 and ZnO particles

for the degradation of undesirable pharmaceutical compounds in water. Detailed insights into

the

potential

applications

of

S-layer

protein-supported nanoparticles are provided in

Sects. 11.5.2 and 11.5.3.



11.3.5 S-Layer Proteins as Filter

Materials

Because of their distinctive characteristics,

S-layer proteins also have potential as filter materials. First, they can easily be applied and aligned

to many surfaces as a result of their selfassembling behavior at phase boundaries.

Second, their well-defined and uniform pore

sizes make them ideal as special micro- or nanosieves with a relatively sharp exclusion limit for

molecules of 30–40 kDa. When combined with

commercial porous support materials and crosslinked with glutaraldehyde, they are known as

S-layer ultrafiltration membranes (SUM) [199,

202].

The surface properties of SUMs can also be

modulated by additional biochemical modificaFig. 11.5 Scanning

electron micrograph of a

bio-composite of the

S-layer of Lysinibacillus

sphaericus JG-A12 and

as-synthesized magnetite

nanoparticles by means

of a one-pot reaction

[157]



J. Raff et al.



tions to optimize the filtration properties of different substances, such as proteins, as well as to

prevent nonspecific adsorption and biofouling

[202]. In short, the selective binding capabilities

of S-layer proteins for different ions embody a

unique selling point for S-layer protein-based

bio-composite materials for both the removal of

toxic elements, as well as for the recovery of

valuable materials from aqueous media (see Sect.

11.5.1). Furthermore, incorporating additional

poly-histidine tags can significantly increase the

nickel-binding capacity of S-layers [139].

Another promising strategy is the combination of

S-layer proteins with metal-binding nanoparticles. When the sponge-like bio-composite from

the S-layer of Lysinibacillus sphaericus JG-A12

is combined with as-synthesized magnetite

nanoparticles (Fig. 11.5), the amount of captured

arsenic (V) could double compared to the binding capacity of the individual components alone

[157].

Thus, by introducing specific metal-binding

motifs through genetic modification and/or coupling of reactive nanoparticles, a wide range of

possibilities to tune S-layer protein-based filtration materials for customer-specific requirements opens up. For example, it should be

possible to separate and concentrate desired

recyclable materials from complex aqueous

streams.



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S-Layer-Based Nanocomposites for Industrial Applications



11.4



S-Layer-Based Coatings

and Their Production



As mentioned in Sect. 11.1, all S-layer proteins

are intrinsically able to spontaneously selfassemble, thereby forming a protein lattice in

suspension, on surfaces, or at interfaces (e.g. air/

water interface) [147]. However, since the complete assembly process is still not fully understood, not all S-layers are easy to recrystallize in

vitro. The self-assembly process is also impacted

by the role of favorable protein concentration and

different bivalent cations such as Ca2+ and Mg2+.

In many cases, polymer formation in suspension

and on surfaces is triggered by the presence of

Ca2+ [7, 91, 190, 210]. Additionally, the surface

properties of coatings are of major importance

for protein adsorption. Typically, most S-layers

possess a negatively-charged inner surface with a

rough topology, whereas the outer surface is

nearly smooth and uncharged. Thus, polarity and

the density of surface charges on the targeted

S-layer influence speed and the covering ratio

during the recrystallization process. For example,

layer formation on hydrophobic surfaces is much

faster compared to analogous hydrophilic-based

processes [125]. Additionally, the S-layer protein

SbpA forms monolayers on hydrophobic surfaces and double layers on hydrophilic silicon

supports [59, 125]. These differences necessitate

a well-considered strategy for modifying the surface properties of a solid support so as to enable

careful control over the S-layer assembling process [30, 59, 197].

Various attempts have been made to investigate the adsorption and self-assembly processes.

For example, researchers have used AFM in a

native environment to visualize proteins [56, 59,

225]. Later, cryogenic TEM was utilized to determine conformational changes during the assembly of S-layer tetramers at the growing boundary

[30]. The recrystallization kinetics on surfaces

have been monitored by quartz crystal

microbalance, leading to several similar explanatory approaches about the steps involved in the

adsorption and assembly of S-layer proteins in

vitro. It should be noted, however, that researchers have utilized different surfaces as a starting



255



point for their investigations, making comparisons difficult. For instance, the [38] used lipid

bilayers; the Sleytr group employed plain mica,

plain silicon supports (hydrophobic, non-plasmatreated and hydrophilic) [59], and silicon polyelectrolyte coated surfaces [213]; while others

used lipid molecules within lipid bilayers, as well

as planar lipid membranes or liposomes [182,

183]. Additionally, the Pollmann group utilized

plain or polyelectrolyte-coated silicon dioxide

[207] and polyelectrolyte hollow spheres [205].

The reassembly of isolated S-layer subunits at

air/water interfaces and on Langmuir-films has

been performed easily and reproducibly at large

scales [203]. In accordance with S-layer proteins

coated on different solid surfaces, the orientation

of the protein arrays at the investigated liquid

interfaces is determined by the anisotropy of the

physicochemical surface properties of the protein

lattice. For example, the protein subunits of

Bacillus coagulans E38-66 are associated with

their more hydrophobic outer face with the air/

water interface, and oriented with their negativelycharged inner face to the zwitterionic head groups

of the lipid monolayer films [152].

In their investigation of another S-layer coating, [98] recrystallized isolated S-layer oblique

(p2) subunits from Bacillus coagulans E38-66 on

positively-charged liposomes. The protein subunits were attached by their negatively charged

inner face in an identical orientation similar to

naturally-occurring cells. By crosslinking the

adsorbed protein layer, additional macromolecules could be attached. Coatings that incorporate S-layer proteins enhance liposome stability

and facilitate additional surface modifications for

targeted applications (e.g., drug delivery) [98].

Also working with Bacillus coagulans E38-66,

[146] demonstrated the ability to recrystallize

S-layer proteins of this strain into closed monolayers at the air/water interface and on solid surfaces pretreated with poly(L-lysine). The S-layer

proteins of this strain were bound to the poly-Llysine coated surfaces via their more hydrophobic and neutral outer face [146].

The Sleytr group also investigated S-layer

coatings on polyelectrolyte surfaces and hollow

spheres; by combining these materials, a novel



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robust biomimetic surface was generated. By

recrystallizing isolated S-layers (SbpA) from

Lysinibacillus sphaericus CCM2177 on polyelectrolyte multilayers, they determined that the

composition of the underlying polyelectrolyte

multilayer plays a crucial role in the structure of

the resulting supported protein layers. On flat

polyelectrolyte-modified surfaces, a crystalline

protein lattice structure was observed equal to

those found in vivo on bacterial cell surfaces.

Importantly, enhanced mechanical stability of the

protein monomer layer was achieved in comparison to surfaces lacking polyelectrolyte modification. Similarly, polyelectrolyte hollow spheres

have also been coated by various techniques

[213], with results confirming that electrostatic

interactions of divalent cations are important for

the self-assembly of S-layer proteins.

Chung et al. [28] utilized in situ atomic force

microscopy to follow the 2D assembly of S-layer

proteins on supported lipid bilayers; their resulting molecular-scaled pictures of Lysinibacillus

sphaericus ATCC 4525 corroborated the multistage pathway of protein recrystallization from

S-layer proteins in the presence of calcium.

Specifically, Chung et al. described the S-layer

protein assembly as a four-step pathway beginning with the adsorption of S-layer monomers

that adsorb onto the lipids in an extended conformation to form amorphous or liquid-like clusters

on the surface. This process is followed by the

condensation of the amorphous cluster, which

then results in the relaxation to the crystalline

nucleus. Finally, self-catalyzed crystal growth

begins with the addition of new tetramers to the

lattice edge sites. Even though ongoing studies

are adding important insights to the recrystallization of S-layer proteins, the detailed mechanisms

associated with protein assembly are still not

fully understood.

Whitelam et al. [225] described protein assembly as a three-phase process: (a) protein aggregation resulting in amorphous clusters on the

membrane, (b) subsequent crystallization of the

protein clusters, and (c) growth via the addition

of further tetramers at the cluster edges.

Subsequently, Pum et al. [151] suggested a more

complex process that is dependent on surface



J. Raff et al.



composition—for example, in the case of mica

and its metastable early crystal clusters.

Bobeth et al. [15] also studied tube formation

during the self-assembly of S-layer-proteins in

order to describe the underlying basic mechanisms and the effects of process parameters on,

for instance, growth velocity and tube radius. The

researchers reported that the initial monomer

concentration is a crucial parameter in the recrystallization process. Specifically, they observed

that a high concentration of monomer leads to

faster growth, which then hinders the arrangement of monocrystalline S-layer patches. A high

patch nucleation rate also results in the growth of

many small assemblates. Bobeth et al. [15] also

discussed how the presence and concentration of

mono- and bivalent- metal cations impacts the

recrystallization process and protein patch formation in their investigated S-layer proteins (e.g.

Lysinibacillus sphaericus NCTC 9602), which

they then compared to other S-layer types

described in the literature.

Our own studies show similar results. As an

example, the recrystallization process of the

S-layer protein SlfB from Lysinibacillus sphaericus JG-A12 on a polyelectrolyte-modified glass

surface is depicted in Fig. 11.6 as AFM images,

as well as in Fig. 11.7 [56]. Over the course of the

process, protein monomers and oligomers adsorb

at the surface and some—but not all—start to

grow. The growth of crystallites on

polyelectrolyte-coated surfaces begins at random

nucleation points (Fig. 11.6, blue arrows) and

proceeds laterally in all directions. Although

growing crystallites can either retain or eliminate

smaller protein aggregates, they do not overgrow

smaller aggregates. Crystal growth proceeds until

the surface is covered by a monolayer of crystalline S-layer protein domains.

QCM-D adsorption and AFM studies of

polyelectrolyte-modified surfaces reveal that

there is no, or only very limited, influence of the

support surface below the polyelectrolyte layers

during the recrystallization process—even in

cases when only one layer of polyelectrolyte is

present. However, the protein recrystallization

process on the support surface is strongly influenced by the charge of the upper polyelectrolyte



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S-Layer-Based Nanocomposites for Industrial Applications



257



Fig. 11.6 AFM images of proceeding recrystallization of

SlfB of Lysinibacillus sphaericus JG-A12 on a

polyelectrolyte-modified glass surface. The red arrows



mark small protein agglomerates and the blue arrows

mark growing crystalline protein patches. (a) after 3.5 h,

(b) after 7.5 h and (c) after 9.25 h



Fig. 11.7 Simplified model of S-layer recrystallization

onto surfaces. (a) Attachment of protein monomers and

oligomers, (b) attachment of further protein monomers to

crystal nucleus leads to a growing protein surface, (c)



growing protein patches and partial integration of small

agglomerates, (d) recrystallization ends after edge contact

of different protein patches. The white arrows show the

direction of the protein patch growth



layer. While anionic and cationic polyelectrolyte

coatings allow the assembly of a closed S-layer

lattice, the recrystallization kinetics differ significantly. Because of the lower affinity of the S-layer

protein to the anionic coating, the crystallization

process is much slower compared to cationic

coatings, thus offering the possibility of

visualizing the different steps via AFM (Fig.

11.6) [56]. Additionally, Suhr et al. [207] confirmed the positive effects on coating kinetics and

protein layer stability by using a polyelectrolyte

as a support layer [206, 207]. In their work, the

authors demonstrated that the S-layer protein

Slp1 from Lysinibacillus sphaericus JG-B53 can

be recrystallized on a polyelectrolyte multilayer

with a positively-charged surface.

Figure 11.8 depicts AFM images of an S-layer

protein coating on a polyelectrolyte-modified



SiO2 surface as recently described by the method

of Günther and Suhr [56, 206, 207]. These images

(amplitude and height image) show the resulting

squared protein lattice of Slp1 from Lysinibacillus

sphaericus JG-B53.

Furthermore, our own studies of S-layer coatings on non-flat substrates reinforce the work of

the Sleytr group in that we used polyelectrolyte

hollow spheres as a template for protein coating,

which was also discussed by Toca-Herrera et al.

[213]. The recrystallization of S-layer proteins on

the outer surface of pre-synthesized polyelectrolyte hollow spheres and modified magnetic polyelectrolyte hollow spheres is shown in Fig. 11.9a.

By using different or multiple types of S-layer

proteins, one can develop an improved bio-based

template for selective metal filter materials, as

well as for the deposition of nanoparticles or the



258



J. Raff et al.



Fig. 11.8 AFM images

of recrystallized S-layer

proteins of

Lysinibacillus

sphaericus JG-B53

(Slp1) on modified

surfaces; (a) height

image and (b) amplitude

image



Fig. 11.9 (a) and (b) Computer-based visualizations of

single and multiple bacterial surface layer proteins coated

on synthesized polyelectrolyte hollow spheres and magnetic hollow spheres for applied research of bio-based

metal and particle templates (Source: S. Münster, HZDR);



(c) REM image of dried polyelectrolyte hollow spheres

coated with S-Layer proteins of Lysinibacillus sphaericus

JG-B53 and adsorbed Pd nanoparticles; (d) corresponding

EDX spectrum of image (c) showing the adsorbed Pd (red

arrows) on the hollow sphere surface [205]



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S-Layer-Based Nanocomposites for Industrial Applications



design of catalytic micro-capsules (Fig. 11.9b).

Also depicted in Fig. 11.9c–d) is the ability to

design 7 μm-sized polyelectrolyte hollow spheres

coated with S-Layer proteins of Lysinibacillus

sphaericus JG-B53 ([205]; unpublished results).

Specifically, Fig. 11.9c shows dried polyelectrolyte hollow spheres coated with S-layer proteins

and adsorbed Pd(0) NP in the protein lattice. The

corresponding EDX spectrum (Fig. 11.9d)

reveals the presence of Pd(0) on the novel

designed biomaterial.

In summary, these examples verify that the

S-layer recrystallization process depends on the

type of solid surface used, as well as modification

and surface properties—e.g., charging and hydrophobicity. In fact, surface characteristics can be

regarded as the most important properties that

influence the recrystallization of S-layer proteins,

their orientation (attached by inner or outer face),

their lattice stability, and the generation of an

almost fully-covered surface. While this information is essential, the fact remains that the mechanisms associated with the recrystallization

process are not completely understood. Thus,

additional research must be undertaken to elucidate the roles of S-layer proteins and their array

formation on solid surfaces and air/water

interfaces.



11.5



New S-Layer-Based

Nanomaterials



259



from titration experiments, certain S-layers proteins bind specifically with Ca2+, thereby forming

very stable complexes (unpublished results).

Based on preliminary evidence, there are at least

two different binding sites for these bivalent cations, which call for further research to determine

their role in polymer formation and proteinprotein interactions.

Second, in addition to the biochemical role of

metal binding, we know that specific metals tend

to bind on the outer and inner surface of the

S-Layer-protein, as well as in its pores, based on

the influence of certain functional groups such as

COOH−, NH2−, OH−, PO4−, SO4− and SO−

(Fig. 11.10). Both the composition and amount of

these functional groups represent characteristic

“fingerprints” of each particular S-Layer-protein,

depending on ionic strength and pH. In this way,

S-layers can serve as ion traps, thereby preventing the uptake of toxic metals into the bacterial

cell.

Researchers have described the importance of

binding metal complexes containing calcium,

strontium, arsenic or antimony with S-layers as

an initial step of bio-mineralization [135, 177,

178]. For example mineral phases such as gypsum, calcite, celestite and strontianite were

formed in this way on S-layers. In these instances,

the S-layers provide crystallization nuclei and

serve as a biomineralization template. Wang &

Müller [221] also discussed the involvement of

S-layers in the formation of polymetallic nodules. Keeping in mind that S-layers are the outer-



11.5.1 Metal/Metalloid Binding

by S-Layers and Their

Applications

Before discussing the potential of S-layer-based

filter materials for either the removal or recovery

of dissolved toxic or valuable metals, two fundamental aspects of the interaction of metals with

S-layer proteins must be discussed. First, some

metals are simply inherently appropriate for the

protein conformation of the single S-layer protein monomer, their polymerization processes,

and the formation of highly-ordered lattices.

These particular metals are bivalent cations such

as Ca2+ and Mg2+ [7, 91, 190, 210]. As known



Fig. 11.10 Schematic drawing of an S-layer monomer

(left) and a tetragonal unit cell (right), including specific

binding sites (symbolized by red circles) and possible

superficial chemical groups of S-layer proteins (unpublished results)



260



J. Raff et al.



most cell component connecting the cell with its

environment, they have to be permeable for all

essential micro- and macro-elements that are fundamental to life. In contrast, their meshwork

structure and high content of free and charged

functional groups (see also Chap. 1) make them

an ideal barrier against toxic dissolved metal

ions, thereby protecting the cell from serious

damage. While it is true that we do not fully

understand how the S-layer discriminates

between essential and toxic metals, this function

has been demonstrated for some bacterial isolates

recovered from a uranium mining waste pile.

Indeed, researchers have described how these

isolates are able to selectively bind toxic metals

such as uranium [117, 158] and arsenic [157]. As

shown in Fig. 11.11, most S-layers have a higher

binding capacity for toxic elements such as uranium or arsenic compared to intact cells. This

fact is really quite surprising given the high density of charged groups in any kind of bacterial

cell wall.

In addition to uranium and arsenic, studies

show that different S-layer proteins can bind several other heavy metals in large amounts. For



example, Shenton et al [188] described the interaction of S-layer proteins with Cd prior to the

formation of CdS nanoparticles [188]. Other

studies include the following: the binding of different precious metals like Pd, Ag, Pt and Au [39,

51, 77, 118, 119, 145, 153, 207, 218]; and the

binding of other metals such as Cr, Ni, Cu and Eu

[26, 145, 158, 207]. As some of these metals

were bound in the range of mg to g per g S-layer

protein on the protein surface, their use for the

production of S-layer-based filtration materials is

obvious. Therefore, it makes more sense to utilize purified S-layer proteins since they are more

specific for a number of metals—in comparison

to a highly complex biomass of intact cells having less specific binding properties. For the

industrial use of S-layer-based filter materials, it

would be essential to generate a large volume of

these proteins in order to ensure cost efficiency.

Such production goals could be achieved by

selecting the ideal S-layer protein and then optimizing isolation procedures, as well as via the

heterologous expression of the protein in host

cells (see also Chaps. 2 and 3).



Fig. 11.11 Arsenic and uranium binding of cells and

S-layers of several isolates recovered from a uranium mining waste pile nearby Johanngeorgenstadt (JG) and of several reference strains. The isolates JG-B62, JG-B53,

JG-B7, JG-A12 and the reference strain NCTC 9602 are

Lysinibacillus sphaericus strains, JG-B58 is a Viridibacillus

arvi strain and all other isolates are different Bacillus spe-



cies. Used reference strains are Geobacillus stearothermophilus DSMZ 13240, Geobacillus stearothermophilus

ATCC 12980, Lysinibacillus fusiformis DSMZ 2898 and

Thermoanaerobacterium

thermosulfurigenes

EM1.

Experiments were carried out with 200 mg U/L for cells

and with 200 mg U/L for S-layers in 0.9 % NaClO4 at pH

4.5 for 48 h or with 10 mg As(V)/L at pH 6.0 for 72 h



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S-Layer-Based Nanocomposites for Industrial Applications



An important use for S-layer-based filter

materials targets the effective removal of toxic

heavy metals such as uranium, and/or toxic metalloids such as arsenic (Fig. 11.11). Even though

S-layer-based materials are more environmentally

friendly and comparably efficient when judged

against polymeric ion exchange materials, their

higher costs currently hamper the wider use of

S-layer-based materials for this purpose. In contrast, their use for the recovery of precious metals

such as Au and Pd is becoming technically and

economically more feasible. Although the binding process occurs quite rapidly [207], as well as

facilitates the recovery of metals from complex

matrices such as highly-saline solutions or matrices containing organic solvents (unpublished

results), the desorption of metals from the protein

bio-composite that would enable their reuse

remains a challenge. Currently, the recovery of

bound precious metals is only possible via thermal decomposition (fuming), which is also carried out for corresponding ion exchange

materials.

Several proteins have been confirmed as

strong calcium binders and, in fact, possess

numerous specific binding sites for calcium. For

example, the Ca-binding protein calmodulin possess EF-hand motifs that bind calcium [35, 127].

In contrast, no calcium-binding motif has yet

been identified for S-layer proteins. Nevertheless,

their binding sites do permit the selective binding

of calcium and chemical-equal elements with

high affinity. These elements include the trivalent

lanthanides, possessing comparable ionic radii

but higher charge densities, which lead to the formation of even more stable metal-protein complexes compared to the corresponding Ca-protein

complexes [220]. One such metal, for example, is

europium. Like many other lanthanides, europium is an industrially important metal that is

used in the production of fluorescent lamps,

light-emitting diodes and many other types of

electronic displays.

In general, it is quite challenging to extract

and separate rare earth metals (principally lanthanides) because so many of them are analogous

in composition, as well as finely distributed in



261



ores. Similarly, the increasingly important need

to recycle electronic products in terms of their

elemental components is hampered by the same

issues. However, one promising approach in

hydrometallurgy is the use of calcium-binding

proteins. For example, S-layer proteins are capable of binding Eu via Ca-binding sites (unpublished results). As a result, these proteins are

suitable for the recovery of at least some lanthanides from natural or industrial water that contain low amounts of trivalent metals and high

amounts of other mono and bivalent metals (e.g.,

K, Na, Ca, Mg) or heavy metals such as Fe, Co or

Ni. In short, some possible applications for

S-layer proteins include the recovery of rare earth

elements from natural water, wastewater from

mining operations, or wastewater in connection

with the recycling of electrical and electronic

equipment. It should also be noted that, in contrast to precious metals, the recovery of a bound

lanthanide is possible by a pH shift, complexing

agents, or elution with buffers possessing high

salinity.

Fundamental for the cost-effective use of

S-layer proteins as a metal-selective binding

matrix is their stable immobilization. Equally

important, therefore, is the preservation of the

native function of the protein polymer during and

after immobilization. Currently, two different

strategies are used to achieve this goal. One possibility is to embed the isolated or cell-bound

protein matrix in a stable, inert and highly porous

SiO2 matrix, which can be achieved via sol–gel

techniques and the production of bio-composite

bulk materials or sol–gel coatings [19, 26, 158].

A second possibility is to coat all industrial carriers with S-layer proteins using a layer-by-layer

technique (see Chap. 4). This latter approach

allows one to coat porous minerals or polymer

bulk materials possessing high surface areas, as

well as polymeric, metallic or ceramic filter

membranes.

In addition to the filtration applications discussed in this section, metal binding with S-layer

proteins is also an important initial step for the

S-layer-mediated production of highly regular

nanostructures, as detailed in Sect. 5.2.



262



11.5.2 S-Layers as Templates

for the Production

of Nanostructures

The defined deposition of regularly structured

metallic (e.g., gold, silver, palladium or iron) and

metal oxide (e.g., zinc oxide, titanium dioxide,

iron oxide) nanoparticles (NPs) is a rapidlygrowing field of research. Recent investigations

of inorganic NPs on the nanoscale range of

approximately 1–100 nm includes studies of their

size control and strategies for assembling them

into well-defined nanostructures on molecular

building blocks [80, 159, 160]. Size- and shapetunable two-dimensional (2D) NP arrays are

attractive for the development of materials with a

variety of physical, optoelectronic, magnetic or

catalytic properties [36, 54, 115, 137, 216, 224].

Additionally, researchers have described the use

of highly-ordered 2D-arrays of metallic or metal

oxide NPs for a variety of bioengineered and biotechnological applications in the fields of (a)

nanoelectronics, (b) biodiagnostics [159], (c)

catalysis [36], (d) photocatalysis [124, 174], and

(e) biosensors [60, 110, 112].

For the development of novel materials, inorganic NPs have to be regularly assembled on

diverse biological entities and structures like

DNA strands, peptides and polymers. The biomolecules can organize the NPs through covalent

and non-covalent interactions [86]. Additionally,



Fig. 11.12 Computer-based reconstructed S-layer lattice

with assembled inorganic NPs in protein pores (Source:

S. Münster/HZDR)



J. Raff et al.



S-layers can be used as scaffolds for the assembly of metallic or metal oxide NPs (Fig. 11.12)

[10, 202]. Recent studies have demonstrated that

the stable periodic structure of S-layer templates

can be used for metal or NP deposition on solid

surfaces in the absence of electricity (electroless

plating) [99, 187]. Due to a wide range of attractive physicochemical properties (e.g., intrinsic

chemical reduction), they are extremely suitable

as templates for the nanofabrication of highlyordered NP arrays. Moreover, the nanoarray fabrication properties of S-layers could be improved

via the surface modification of the protein functional groups with different molecules [11, 119,

131, 145, 201, 203]. Therefore, S-layer proteins

represent an alternative (or parallel) approach for

the structural organization of 2D-NP arrays,

especially in comparison to currently used nanolithographic techniques such as electron beam

lithography, scanning probe lithography, chemical synthesis or laser-focused atom deposition

[63, 115]. Importantly, the immobilization of

reactive and nanofabricated NPs into wellordered structures results in many interesting

properties that are unique for the specific assembled NP on S-layer templates [86]. For example,

these deposited, highly-ordered NPs can be used

for materials with high catalytically-active

surfaces.

The current focus for reactive NPs, however,

is on noble metals such as gold (Au) and the platinum group metals, including platinum (Pt), palladium (Pd) and rhodium (Rh)—principally

because of their increased role in catalysis and

for use in electronics applications. Closely related

to this emphasis is the recovery of noble metals—for example from industrial waste or process waters—to address the growing demand for

these resources. Bio-based and designed materials such as metallic NPs on S-layer templates can

be used for the catalysis of diverse chemical reactions like hetero-chemical hydration or carboncarbon coupling reactions.

Based on the natural properties of living bacteria to form minerals in the environment [45,

149], as well as the ability of bacteria and isolated outermost cell components like S-layers to

bind different precious metals in high capacities



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4 Repeat Proteins as Scaffolds for Biomolecular Patterning

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