Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

Podsumowanie

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

Streszczenie

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis ¹ or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers ^2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.

The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Wprowadzenie

Due to the increasing use of gold for several applications like electronics, catalysts, biosensors, or medical instruments, the demand of this precious metal has grown over the last few years' time ^6-9. Gold as well as many other precious and heavy metals are released into the environment via industrial effluents in dilute concentrations, through mining activities, and waste disposal ^7,8,10,although most environmental contamination by heavy or precious metals is an on-going process mainly caused by technological activities. This leads to a significant interference of natural ecosystems and could potentially threaten human health ⁹. Knowing these negative outcomes promotes the search for new techniques to remove metals from contaminated ecosystems and improvements in recycling metals from industrial wastewater. Well-established physico-chemical methods like precipitation or ion exchange are not so effective, especially in highly diluted solutions ^7,8,11. Biosorption, either with living or dead biomass, is an attractive alternative for wastewater treatment ^10,12. The use of such biological materials can reduce the consumption of toxic chemicals. Many microorganisms have been described to accumulate or immobilize metals. For instance, cells of Lysinibacillus sphaericus (L. sphaericus) JG-A12 have shown high binding capacities for precious metals, e.g., Pd(II), Pt(II), Au (III), and other toxic metals like Pb(II) or U(VI) ^4,13, cells of Bacillus megaterium for Cr(VI) ¹⁴, cells of Saccharomyces cerevisiae for Pt(II) and Pd(II) ¹⁵, and Chlorella vulgar for Au(III) and U(VI) ^16,17. The binding of previous metals like Au(III), Pd(II), and Pt(II) has also been reported for Desulfovibrio desulfuricans¹⁸ and for L. sphaericus JG-B53 ^19,20. Nevertheless, not all microbes bind high amounts of metals and their application as sorptive material is limited ^12,21. Furthermore, metal binding capacity depends on different parameters, e.g., cell composition, the used bio-component, or environmental and experimental conditions (pH, ionic strength, temperature etc.). The study of isolated cell wall fragments ^22,23, like membrane lipids, peptidoglycan, proteins, or other components, helps to understand the metal binding processes of complex constructed whole cells ^8,21.

The cell components focused on in this study are S-layer proteins. S-layer proteins are parts of the outer cell envelope of many bacteria and archaea, and they constitute about 15 - 20% of the total protein mass of these organisms. As the first interface to the environment, these cell compounds strongly influence the bacterial sorption properties ³. S-layer proteins with molecular weights ranging from forty to hundreds of kDa are produced within the cell, but are assembled outside where they are able to form layers on the lipid membranes or polymeric cell wall components. Once isolated, nearly all S-layer proteins have the intrinsic property to spontaneously self-assemble in suspension, at interfaces, or on surfaces forming planar or tube-like structures ³. The thickness of the protein monolayer depends on the bacteria and is within a range of 5 - 25 nm ²⁴. In general, the formed S-layer protein structures can have an oblique (p1 or p2), square (p4), or hexagonal (p3 or p6) symmetry with lattice constants of 2.5 to 35 nm ^3,24. The lattice formation seems to be in many cases dependent on divalent cations and mainly on Ca^{2+ 25,26, Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove & Aitziber L. Cortajarena) (Springer, 2016 (submitted))}. Nevertheless, the full reaction cascade of monomer folding, monomer-monomer interaction, the formation of a lattice, and the role of different metals, especially of divalent cations such as Ca²⁺ and Mg²⁺, are still not fully understood.

The gram-positive strain L. sphaericus JG-B53 (renamed from Bacillus sphaericus after new phylogenetic classification) ²⁷ was isolated from the uranium mining waste pile "Haberland" (Johanngeorgenstadt, Saxony, Germany) ^4,28,29. Its functional S-layer protein (Slp1) possesses a square lattice, a molecular weight of 116 kDa ³⁰, and a thickness of ≈ 10 nm on living bacteria cells ³¹. In previous studies, the in vitro formation of a closed and stable protein layer with a thickness of approximately 10 nm was achieved in less than 10 min ¹⁹. The related strain L. sphaericus JG-A12, also an isolate from the "Haberland" pile, possesses high metal binding capacities and its isolated S-layer protein has shown a high chemical and mechanical stability and good sorption rates for precious metals like Au(III), Pt(II), and Pd(II) ^4,32,33. This binding of precious metals is more or less specific for some metals and depends on the availability of functional groups on the outer and inner protein surface of the polymer and in its pores, ionic strength, and the pH value. Relevant functional groups for metal interaction by the proteins are COOH-, NH₂-, OH-, PO₄-, SO₄-, and SO-. In principle, metal binding capacities open a wide spectrum of applications,^{Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove & Aitziber L. Cortajarena) (Springer, 2016 (submitted)).}e.g., as biosorptive components for removal or recovery of dissolved toxic or valuable metals, templates for synthesis or defined deposition of regularly structured metallic nanoparticles (NPs) for catalysis, and other bio-engineered materials like bio-sensory layers ^3,5,18,33. Regularly arranged NP arrays like Au(0)-NPs could be used for major applications ranging from molecular electronics and biosensors, ultrahigh density storage devices, and catalysts for CO-oxidation ^34-37. The development of such applications and smart design of these materials necessitates a deeper understanding of the underlying metal binding mechanisms.

A prerequisite for the development of such bio-based materials is the reliable implementation of an interface layer between the biomolecule and the technical surface ^38,39. For example, polyelectrolytes assembled with the layer-by-layer (LbL) technique ^40,41 have been used as an interface layer for recrystallization of S-layer proteins ³⁹. Such an interface offers a relatively easy way to perform the protein coating in a reproducible and quantitative way. By performing different experiments with and without modification with adhesive promoters, it is possible to make statements regarding coating kinetics, layer stability, and interaction of metals with biomolecules ^{19,42,Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove & Aitziber L. Cortajarena) (Springer, 2016 (submitted))}. However, the complex mechanism of the protein adsorption and protein-surface interaction is not completely understood. Especially information on conformation, pattern orientation, and coating densities is still missing.

Quartz crystal microbalance with dissipation monitoring (QCM-D) technique has attracted attention in the recent years as a tool for studying protein adsorption, coating kinetics, and interaction processes on the nanometer scale ^19,43-45. This technique allows for the detailed detection of mass adsorption in real-time, and can be used as an indicator for the protein self-assembling process and coupling of functional molecules on protein lattices ^{19,20,42,46-48}. In addition, QCM-D measurements open the possibility to study metal interaction processes with the proteinaceous layer under natural biological conditions. In a recent study, the interaction of the S-layer protein with selected metals like Eu(III), Au(III), Pd(II),and Pt(II) has been studied with QCM-D ^19,20. The adsorbed protein layer can serve as a simplified model of a cell wall of gram-positive bacteria. The study of this single component can contribute to a deeper understanding of metal interaction. However, solely QCM-D experiments do not allow statements regarding surface structures and influences of metals to protein. Other techniques are necessary to obtain such information. One possibility for imaging bio-nanostructures and obtaining information on structural properties is the atomic force microscopy (AFM).

The objective of the presented study was to investigate the sorption of gold (Au(III) and Au(0)-NPs) to S-layer proteins, in particular Slp1 of L. sphaericus JG-B53. Experiments were done with suspended proteins on batch scale in a pH range of 2.0 - 5.0 using ICP-MS and with immobilized S-layers using QCM-D. Additionally, the influence of metal salt solution on the lattice stability was investigated with subsequent AFM studies. The combination of these techniques contributes to a better understanding of in vitro metal interaction processes as a tool for learning more about binding events on whole bacterial cells regarding specific metal affinities. This knowledge is not only crucial for the development of applicable filter materials for the recovery of metals for environmental protection and the conservation of resources ⁴⁹, but also for the development of arrays of highly ordered metallic NPs for various technical applications.

Protokół

1. Microorganism and Cultivation Conditions

Note: All experiments were done under sterile conditions. L. sphaericus JG-B53 was obtained from a cryo-preserved culture ^29,30.

1. Transfer cryo-preserved culture (1.5 ml) under the clean bench to 300 ml sterile nutrient broth (NB) media (3 g/L meat extract, 5 g/L peptone, 10 g/L NaCl). Afterwards stir the solution for at least 6 hr at 30 °C to obtain the pre-culture for cultivation.
2. Cultivate the bacteria under aerobic conditions in NB media at pH = 7.0, 30 °C in a 70 L scaled steam-in-place bioreactor. Therefore, fill the reactor with ≈ 57 L deionized water. Add and dissolve solid NB media directly in bioreactor (concentrations see above).
   1. Additionally add antifoam agent (30 µl/L NB-media) to the media to suppress foam formation during cultivation, then autoclave (122 °C, temperature holding time 30 min) the media inside the reactor facility.
3. Cool down the media and perform complete oxygen saturation. Adjust the pH to 7.0 (using 1 M H₂SO₄ and 2 M NaOH) and start the automatic inoculation of the 300 ml pre-culture. Start the data recording of cultivation parameters at the inoculation point. Log online parameters e.g., dissolved oxygen level (dO₂), acid and base addition, and pH-values within the cultivation.
   1. Monitor the bacterial growth online by the non-invasive turbidity measurements.
4. Perform additional sampling after every hour of cultivation and determine further parameter such as bio dry weight (BDW) and offline optical density (OD). Therefore, collect 20 ml cultivation broth at each sampling point under sterile conditions.
   1. Determine offline OD by photometric measurements of the adsorption at 600 nm. Use sterile filtrated NB-medium as a blank value. After reaching adsorption >0.4 dilute the cell suspension following the linearity of the Lambert-Beer law.
   2. For determination of BDW centrifuge 1 to 5 ml of bacterial suspension (depending on cell density) at 5,000 x g for 5 min at RT. Dry the obtained cell pellet at 105 °C in a heating oven up to mass stability and measure the pellet mass.
5. Take microscopic images with optical phase contrast research microscope in 400 and 1,000 fold magnification (phase contrast condenser 2 and 3 respectively) for checking of the bacterial growth and as a cross-contamination control.
6. After reaching the exponential growth phase detected by online dO₂ and online turbidity, harvest the biomass by flow-through centrifugation at 15,000 x g, 4 °C and wash the biomass twice with standard buffer (50 mM TRIS, 10 mM MgCl₂, 3 mM NaN₃, pH = 7.5).
   Note: The obtained biomass pellet can be stored at -18 °C until further usage for isolation.

2. S-layer Protein Isolation and Purification

Note: Purify Slp1 polymers according to an adapted method as described previously^{2,19,30,32,50,51}.

1. Homogenize the washed and defrosted crude biomass obtained from cultivation in standard buffer (1:1 (w/v)) to remove flagella by using a disperser (level 3, 10 min) under ice bath cooling at 4 °C.
2. Centrifuge the suspension (8,000 x g, 4 °C for 20 min) and wash the obtained pellet twice with standard buffer (1:1 (w/v)). After washing and centrifugation (8,000 x g, 4 °C for 20 min), resuspend the pellet in standard buffer (1:1 (w/v)), add DNase II and RNase (0.4 units/g biomass) to the suspension and disintegrate the cells at 1,000 bar with a high-pressure homogenizer. Afterwards centrifuge the suspension at 27,500 x g, 4 °C for 1 hr.
   Note: Control cell suspension with the research microscope. Rupture is completed when less than 2 - 3 intact cells are visible in the view field of the microscope in 400 fold magnification.
3. Wash the pellet twice with standard buffer (1:1 (w/v)) and perform the centrifugation again. Afterwards resuspend the pellet in standard buffer (2:1 (w/v)) mixed with 1% Triton X-100 and incubate it for 20 min under successive shaking (100 rpm) to solubilize lipid deposits.
4. Centrifuge the solution (27,500 x g, 4 °C for 1 hr) and wash the obtained pellet three times with standard buffer (1:1 (w/v)).
5. Incubate the pellet obtained after additional centrifugation (27,500 x g, 4 °C for 1 hr) for 6 hr in standard buffer (1:1 (w/v)) mixed with 0.2 g/L lysozyme, to hydrolyze linkages in peptidoglycan ⁵⁰. Additionally add DNase II and RNase (each 0.4 units/g biomass) to the suspension.
6. After centrifugation (45,500 x g, 4 °C, 1 hr), resuspend the upper white protein phase with a low volume of the centrifugation supernatant (<30 ml) containing protein subunits.
7. Solubilize the white suspension by mixing 1:1 with 6 M guanidine hydrochloride (6 M GuHCl, 50 mM TRIS, pH = 7.2). The solution becomes bright.
8. Perform sterile filtration (0.2 µm) of the GuHCl treated solution followed by an additional high-speed centrifugation (45,500 x g, 4 °C for 1 hr).
9. Transfer the supernatant to dialysis membrane tubes (MWCO 50,000 Dalton) and dialyzed it against recrystallization buffer (1.5 mM TRIS, 10 mM CaCl₂, pH = 8.0) for 48 hr.
10. Transfer the white recrystallized protein polymer solution into tubes and centrifuge at 45,500 x g, 4 °C for 1 hr. Resuspend the pellet in a low volume of ultrapure water (<30 ml).
11. Afterwards, transfer the suspension into dialysis membrane tubes and perform a dialysis against ultrapure water for 24 hr to remove buffer content.
    Note: Several changes of buffer or ultrapure water during dialysis are indispensable.
12. Lyophilize the purified Slp1 in a freeze dryer.

3. Characterization and Quantification of Slp1 for Experiments

Note: Slp1 concentration for sorption and coating experiments were quantified by UV-VIS spectrophotometry.

1. Pipette 2 µl of dissolved Slp1 sample directly onto the lower measurement pedestal of the photometer. Determine protein concentration at adsorption maximum at a wavelength of 280 nm, characteristic for proteins. Use the extinction coefficient of 0.61 to determine Slp1 concentration. Use Slp1 free solution for reference measurements.
2. Dilute the protein with the buffer (for sorption experiments in batch mode use 0.9% NaCl, pH = 6.0 and for QCM-D experiments use recrystallization buffer, pH = 8.0) to desired concentration for experiments (1 g/L and 0.2 g/L respectively).
3. Analyze Slp1 quality and molecular weight by the standard bioanalytical method sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) described by Laemmli, U. K. ⁵².
   1. Perform SDS-PAGE before using Slp1 within experiments and e.g., after Au(0)-NP incubation using 10% polyacrylamide separation gels.
   2. For SDS-samples mix ≈10 µl of the cultivation or protein sample with sample buffer (1.97 g TRIS, 5 mg bromophenol blue, 5.8 ml glycerin, 1 g SDS, 2.5 ml β-mercaptoethanol, fill with ultrapure water to 50 ml) in a ratio of 1:1 (v/v) and pipette the mixture after 4 min incubation at 95 °C into the gel pockets.
   3. Run SDS-PAGE 30 min at a voltage of 60 V until the samples pass the collection gel and change voltage to 120 V once passing the separation gel.
   4. Remove the gels from the gel system, rinse with ultrapure water and place for 1 hr into fixation solution (10% acidic acid, 50% absolute ethanol). Afterwards, rinse the gels with ultrapure water.
   5. Stain gels by using an adapted unspecific colloidal Coomassie brilliant blue method ^53,54. After destaining^72,73, take SDS-PAGE images by the gel documentation system according to manufacturer's protocol.

4. Sorption Experiments in Batch-mode and Metal Quantification

1. For batch sorption experiments prepare Au(III) stock solution from HAuCl₄ ∙ 3 H₂O, dilute the metal salt and mix it with the Slp1/ NaCl solution to an initial metal concentration of 1 mM and final Slp1 concentration of 1 g/L. Perform experiments in triplets with an additional negative control without Slp1. Use a total volume of 5 ml for sorption experiments.
2. Shake the suspension continuously at RT at different pre-adjusted pH values between 2.0 to 5.0 for 24 hr (adjust pH with low concentrated HCl and NaOH solution).
3. After sorption, centrifuge the samples at 15,000 x g, 4 °C for 20 min) to separate Slp1 from supernatant.
4. Transfer the supernatant into ultrafiltration tubes (MWCO 50,000 Da) and centrifuge this at 15,000 x g, 4 °C for 20 min to remove dissolved protein monomers.
5. Determine the metal concentration in the resulting filtrate by ICP-MS ^19,20 and use the results for back-calculation of sorbed metal by the Slp1 dry mass. Measuring principles, opportunities of the method and components of the used ICP-MS were described in literature ⁵⁵.
   1. Prepare samples and references for ICP-MS measurements using 1% HNO₃ as matrix and rhodium as an internal standard (1 mg/ml).

5. Synthesis of Au-NP and Determination of Particle Size

Note: Citrate stabilized Au(0)-NP were synthesized according to an adapted method described previously by Mühlpfordt, H. et al. (1982) to obtain spherical particles with a diameter of 10 - 15 nm ^56,57.

1. Prepare a stabilized 25 mM HAuCl₄ ∙ 3 H₂O stock for the NP formation.
2. Dilute 250 µl of this stock solution in 19.75 ml ultrapure water and incubate these at 61 °C for 15 min under successive shaking.
3. Prepare 5 ml of a second stock solution (12 mM tannic acid, 7 mM sodium citrate di-hydrate, 0.05 mM K₂CO₃) and incubate the 2^nd solution separately at 61 °C for 15 min.
4. Add under constant stirring the 2^nd stock solution to solution one. Stir the reaction mixture for at least 10 min at 61 °C. Afterwards cool down the solution and use it for NP coating on Slp1 lattice within QCM-D experiments.
   Note: The resulting Au(0)-NP were characterized with UV-VIS spectroscopy at the absorbance maximum of 520 nm, typically used for the detection of formed Au(0)-NPs ⁵⁸. The solution can be stored at 4 °C.
5. Analyze the size of the formed Au(0)-NP by photon correlation spectroscopy (PCS) which is also known as dynamic light scattering.
   1. For determination of NP size, transfer 1.5 ml of synthesized Au(0)-NP solution into cuvettes under dust-free conditions in a laminar flow box and analyze it with a size and zeta potential particle sizer. A detailed description of PCS and sample preparation is given in Schurtenberger, P. et al. (1993) ⁵⁹ and Jain, R. et al. (2015) ⁶⁰.

6. QCM-D Experiments - Slp1 Coating on Surfaces and Au-NP Adsorption onto Slp1 Lattice

Note: Measurements were carried out with a QCM-D equipped with up to four flow modules. All QCM-D experiments were performed with a constant flow rate of 125 µl/min at 25 °C. Slp1 coating and metal/NP incubation were done on SiO₂ piezoelectric AT-cut quartz sensors (Ø 14 mm) with a fundamental frequency of ≈ 5 MHz. Rinsing steps and addition of solution are marked in the figures of the representative results part. The QCM-D experiments could be described as a step by step way beginning with cleaning and surface modification of the used sensors followed by Slp1 recrystallization and later on the metal and metal NP interaction.

1. Cleaning Procedures:
   1. Equip the fluid cells with sensor dummies. Pump at least 20 ml (each per module) of an alkaline liquid cleansing agent (2% cleanser in ultrapure water (v/v)) through the QCM-D and tube system. Afterwards pump the fivefold volume (each per module) of ultrapure water through the system (flow rate up to 300 µl/min). Perform the cleaning according to the manufacturer's protocol.
   2. Clean the SiO₂ sensors outside the flow modules by incubation (at least 20 min) in 2% SDS solution and rinse the sensors afterwards several times with ultrapure water ^61,62.
   3. Dry the crystals with filtered compressed air and place them in an ozone cleaning chamber for 20 min ^63,64.
   4. Repeat the cleaning procedure twice to remove all organic contents.
   5. To remove bound metals from the sensor surface rinse the sensors with 1 M HNO₃. Afterwards, perform all rinsing steps with ultrapure water.
2. Sensor Surface Modification by Polyelectrolytes:
   Note: Surface modification can be done either inside (flow through procedure) or outside the flow module (LbL technique). Within these experiments the following way to modify the surfaces was used.
   1. Modify the sensors with 3 g/L of alternating PE layers of polyethylene imine (PEI, MW 25,000) and polystyrene sulfonate (PSS, MW 70,000) via dip coating using LbL technique ^40,41 described previously for the special used system in the article by Suhr, M. et al. (2014) ¹⁹.
   2. Place the sensors inside the appropriate PE-solution in deep well plates and incubate these for 10 min at RT.
   3. Take the sensors out of PE-solution and rinse the sensors between every dip coating step intensively with ultrapure water.
      Note: The new surface modification consists of at least three PE layer terminating with positively charged PEI.
   4. After this external modification place the sensors inside the flow module and equilibrate the sensors by rinsing with ultrapure water before starting the experiments.
3. Slp1 Monolayer Recrystallization:
   1. Dissolve Slp1 in 4 M urea for converting polymers into monomers.
   2. Centrifuge the monomerized proteins at 15,000 x g, 4 °C for 1 hr to remove bigger protein agglomerates.
   3. Mix the solubilized and centrifuged Slp1 supernatant with recrystallization buffer to a final protein concentration of 0.2 g/L.
      Note: The calcium depending recrystallization of Slp1 (self-assembling) starts by addition of the recrystallization buffer. Therefore, pump the mixed solution with a flow rate of 125 µl/min to the sensors (placed inside the flow modules) immediately. The recrystallization is done after stable values of frequency and dissipation shifts were detected within QCM-D experiments.
   4. After successful protein recrystallization on top of the PE-modified sensors inside the flow modules rinse the coated sensors with recrystallization buffer or ultrapure waterintensively with a flow rate of 125 µl/min until stable values of frequency and dissipation shifts were detected.
      Note: The SiO₂ surface modification with PE for later sorption experiments onto Slp1 monolayer and AFM studies is visualized in Figure 1.

Figure 1. Schematic Design of PE Surface Modification and Slp1 Monolayer Coating; This figure has been modified from Suhr, M. et al. (2015) ¹⁹ with permission from Springer. Please click here to view a larger version of this figure.

4. Metal and Metal NP Interaction:
   Note: The sorption with the Au metal salt solution (HAuCl₄ ∙ 3 H₂O) was carried out in concentrations of 1 mM or 5 mM at pH = 6.0 in 0.9% NaCl solutions. Au-NP adsorption was done with undiluted Au-NPs in 1.6 mM tri-sodium-citrate buffer at pH ≈ 5.0.
   1. After successful Slp1 coating in the flow modules, rinse the obtained Slp1 layer intensively with 0.9% NaCl solution until stable values of frequency and dissipation shifts were detected.
   2. Pump the prepared metal solution (1 mM) and NP solution to the flow modules with a flow rate of 125 µl/min and track the mass adsorption to the Slp1 layer. Mass adsorption can be detected directly by tracking the frequency shifts referring to the Sauerbrey equation (Equation 1).
   3. After completing the metal and metal NP interaction, rinse the layer with metal/NP free buffer to remove weak bound or weak attached metals or nanoparticles.
      Note: An illustration of the experimental setup is shown in Figure 2.

Figure 2. Schematic Design of QCM-D Setup using Flow Module QFM 401*⁶⁶. Please click here to view a larger version of this figure.

5. Data Recording and Evaluation:
   1. Record the shifts in frequency in Hz (Δf_n) and dissipation (ΔD_n) within the QCM-D experiments by using QCM-D specific software.
   2. Use for evaluation of the adsorbed mass sensitivity (Δm) the Sauerbrey equation/model (Equation 1) ^{65 66} that is valid for thin and rigid films coupled without friction to the sensor surface applied to the n^th overtone. The term C (Sauerbrey constant) for the used 5 MHz AT cut quartz sensor is 17.7 ng ∙ Hz^-1 ∙ cm^-268. For rigid, evenly distributed, and sufficiently thin adsorbed layers use Equation 1 as a good approximation.
      
   3. Perform additional modeling according to the Kelvin-Voigt model valid for viscoelastic molecules ^68-71 with the manufacturer specific software and compare the results with that of the Sauerbrey model.
   4. For calculation of the layer thickness and mass adsorption use as important modelling parameter a layer density of the adsorbed layer of 1.35 g ∙ cm^-3 corresponding to values described previously for S-layer proteins ^72-75. Use the same value for the calculation of metal interaction with the proteinaceous layer.

7. AFM Measurements

1. Perform studies with fully capable AFM on an inverted optical microscope.
   1. Record AFM images in liquid using the recrystallization buffer or ultrapure water directly on the coated QCM-D sensors.
   2. Rinse the sensors with ultrapure water after QCM-D experiments and place them inside the AFM fluid cell. Therefore, use a closed fluid cell with a total volume of about 1.5 ml. Keep the temperature of the fluid cell constant at 30 °C.
   3. Use a cantilever with a resonance frequency of ≈ 25 kHz in water and a stiffness of <0.1 N/m. Adjust the scanning speed between 2.5 and 10 µm/sec.
   4. Take images in dynamic contact mode while the cantilever is excited by a piezo at its resonance frequency. Determine the distance of the cantilever to the surface by the oscillation damping ⁷⁶.
      Note: Height images are shown with z-scale while z-values represent the exact topography of the surface. Amplitude (Pseudo 3D) images are shown without z-scale because amplitude z-values depend on scanning parameters and bear limited information. Analysis of images was done using three different evaluation software's ⁷⁷.

Wyniki

Cultivation of Microorganisms and Slp1 Characterization

The recorded data of bacterial growth indicates the end of the exponential growth phase at around 5 hr. Previous investigations have shown that Slp1 can be isolated from this point of harvest (4.36 g/L wet biomass (≈ 1.45 g/L (BDW)) with a maximum yield ¹⁹. Nevertheless, optimization of cultivation by using defined media components or fed-b...

Dyskusje

In this work studied the binding of Au to S-layer proteins was investigated using a combination of different analytical methods. In particular, the binding of Au is very attractive not only for the recovery of Au from mining waters or process solutions, but also for the construction of materials, e.g., sensory surfaces. For studies of the Au interaction (Au(III) and Au(0)-NPs) with suspended and recrystallized monolayer of Slp1, the protein had to be isolated. Therefore, this study has shown the successful culti...

Materiały

Name	Company	Catalog Number	Comments
equiment and software
Bioreactor, Steam In Place 70L Pilot System	Applikon Biotechnology, Netherlands	Z6X	Including dO2, pH sensors of Applikon Biotechnology and BioXpert software V2
Noninvasive Biomass Monitor BugEye 2100	BugLab, Concord (CA), USA	Z9X	---
Spectrometer Ultrospec 1000	Amersham Pharmacia Biotech, Great Britain	80-2109-10	Company now GE Healthcare Life Sciences
MiniStar micro centrifuge	VWR, Germany	521-2844	For centrifugation of cultivation samples
Research system microscope BX-61	Olympus Germany LLC, Germany	037006	Microscope in combination with imaging software
Cell^P (version 3.1)	Olympus Soft Imaging Solutions LLC, Münster, Germany	---	together with microscope
Powerfuge Pilot Separation System Serie 9010-S	Carr Centritech, Florida, USA	9010PLT	For biomasse harvesting
T18 basic Ultra Turrax	IKA Labortechnik, Germany	431-2601	For flagella removal and sample homogenization
Sorvall Evolution RC Superspeed Centrifuge	Thermo Fisher Scientific, USA	728411	Used within protein isolation
Mobile high shear fluid processor, M-110EH-30 Pilot	Microfluidics, Massachusetts, USA	M110EH30K	Used for cell rupture
Alpha 1-4 LSC Freeze dryer	Martin Christ Freeze dryers LLC, Osterode, Germany	102041	---
UV-VIS spectrophotometry (NanoDrop 2000c)	Thermo Fisher Scientific, USA	91-ND-2000C-L	For determination of protein concentration
Mini-PROTEAN vertical electrophoresis chamber	Bio-Rad Laboratories GmbH, Munich, Germany	165-3322	For SDS-PAGE
VersaDoc Imaging System 3000	Bio-Rad Laboratories GmbH, Munich, Germany	1708030	Used for imaging of SDS-PAGE gels
ICP-MS Elan 9000	PerkinElmer, Waltham (MA), USA	N8120536	For determination of metal concentration
Zetasizer Nano ZS	Malvern Instruments, Worcestershire United Kingdom	ZEN3600	For determination of nanoparticle size
Q-Sense E4 device	Q-Sense AB, Gothenburg, Sweden	QS-E4	ordered via LOT quantum design (software included with E4 platform)
Q-Soft 401 (data recording)	Q-Sense AB, Gothenburg, Sweden
Q-Tools 3 (data evaluation and modelling)	Q-Sense AB, Gothenburg, Sweden
QCM-D flow modules QFM 401	Q-Sense AB, Gothenburg, Sweden	QS-QFM401	ordered via LOT quantum design
QSX 303 SiO2 piezoelectric AT-cut quartz sensors	Q-Sense AB, Gothenburg, Sweden	QS-QSX303	ordered via LOT quantum design
Ozone cleaning chamber	Bioforce Nanoscience, Ames (IA), USA	QS-ESA006	ordered via LOT quantum design
Atomic Force Microscope MFP-3D Bio AFM	Asylum Research, Santa Barbara (CA), USA	MFP-3DBio	AFM measurements and imaging software
Asylum Research AFM Software AR Version 120804+1223	Asylum Research, Santa Barbara (CA), USA	---	imaging software included in Cat. No. MFP-3DBio
Igor Version Pro 6.3.2.3 Software	WaveMetrics, Inc., USA	---	imaging software included in Cat. No. MFP-3DBio
BioHeater	Asylum Research, Santa Barbara (CA), USA	Bioheater	Sample heater for AFM measurements
Biolever mini cantilever,  BL-AC40TS-C2	Olympus Germany LLC, Germany	BL-AC40TS-C2	Prefered cantilever for AFM measurements
WSxM 5.0 Develop 6.5 (2013)	Nanotec Electronica S.L. , Spain	freeware	Software for AFM analysis
Name	Company	Catalog Number	Comments
Detergents and other equiment
Calcium chloride Dihydrate (CaCl2 ∙ 2H2O)	Merck KGaA	1.02382	---
acidic acid, 100 %, p.A.	CARL ROTH GmbH+CO.KG	3738.5	Danger, flammable and corrosive liquid and vapour. Causes severe skin burns and eye damage.
Antifoam 204	Sigma-Aldrich Co. LLC.	A6426	For foam suppression
bromophenol blue, sodium salt	Sigma-Aldrich Co. LLC.	B5525	---
Coomassie Brilliant Blue R (C45H44N3NaO7S2)	CARL ROTH GmbH+CO.KG	3862.1	---
Deoxyribonuclease II from porcine spleen	Sigma-Aldrich Co. LLC.	D4138	Typ IV , 2,000-6,000 Kunitz units/mg protein
Ethanol, 95%	VWR, Germany	20827.467	Danger, flammable
glycerine, p.A.	CARL ROTH GmbH+CO.KG	3783.1	---
Gold(III) chloride trihydrate (HAuCl4 ∙ 3H2O)	Sigma-Aldrich Co. LLC.	520918	Danger
Guanidine hydrochloride (GuHCl)	CARL ROTH GmbH+CO.KG	0037.1	---
Hellmanex III	Hellma GmbH & Co. KG	9-307-011-4-507	---
Hydrochloric acid (HCl) (37%)	CARL ROTH GmbH+CO.KG	4625.2	Danger; Corrosive, used for pH adjustment
Lysozyme from chicken egg white	Sigma-Aldrich Co. LLC.	L6876	Lyophilized powder, protein =90 %, =40,000 units/mg protein (Sigma)
Magnesium chloride Hexahydrate (MgCl2 ∙ 6H2O)	Merck KGaA	1.05833	---
Magnetic stirrer with heating,  MR 3000K	Heidolph Instruments GmbH & Co.KG, Germany	504.10100.00	Standard stirrer within experiment
NB-Media DM180	Mast Diagnostica GmbH	121800	---
Nitric acid (HNO3)	CARL ROTH GmbH+CO.KG	HN50.1	Danger; Oxidizing, Corrosing
PageRuler Unstained Protein Ladder	ThermoScientific-Pierce	26614	---
Poly(sodium 4-styrenesulfonat) (PSS)	Sigma-Aldrich Co. LLC.	243051	Average Mw ~70,000
Polyethylenimine (PEI), branched	Sigma-Aldrich Co. LLC.	408727	Warning; Harmful, Irritant, Dangerous for the environment; average Mw ~25,000
Potassium carbonate anhydrous (K2CO3)	Sigma-Aldrich Co. LLC.	60108	Warning; Harmful
Ribonuclease A from bovine pancreas	Sigma-Aldrich Co. LLC.	R5503	Type I-AS, 50-100 Kunitz units/mg protein
Sodium azide (NaN3)	Merck KGaA	106688	Danger; very toxic and Dangerous for the environment
Sodium chloride (NaCl)	CARL ROTH GmbH+CO.KG	3957.2	---
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich Co. LLC.	L-5750	Danger; toxic
Sodium hydroxide (NaOH)	CARL ROTH GmbH+CO.KG	6771.1	Danger; Corrosive, used for pH regulation within cultivation and pH adjustment
Spectra/Por 6, Dialysis membrane, MWCO 50,000	CARL ROTH GmbH+CO.KG	1893.1	---
Sulfuric acid (H2SO4)	CARL ROTH GmbH+CO.KG	HN52.2	Danger; Corrosive, used for pH regulation within cultivation
Tannic acid (C76H52O46)	Sigma-Aldrich Co. LLC.	16201	---
TRIS HCl (C4H11NO3HCl)	CARL ROTH GmbH+CO.KG	9090.2	---
Tri-sodium citrate dihydrate (C6H5Na3O7 ∙ 2H2O)	CARL ROTH GmbH+CO.KG	3580.2	---
Triton X-100	CARL ROTH GmbH+CO.KG	3051.3	Warning; Harmful, Dangerous for the environment
VIVASPIN 500, 50.000 MWCO Ultrafiltration tubes	Sartorius AG	VS0132	---
β-mercaptoethanol	Sigma-Aldrich Co. LLC.	M6250	Danger, toxic