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Catalytically Active Protein Coatings: Towards Enzymatic Cascade Reactions at Inter-Colloidal Level Max Julius Maennel, Lucas Philipp Kreuzer, Christian Goldhahn, Jonas Schubert, Maximilian Johannes Hartl, and Munish Chanana ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03072 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017
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Catalytically Active Protein Coatings: Towards Enzymatic Cascade Reactions at Inter-Colloidal Level Max Julius Männel, 2,4 Lucas Philipp Kreuzer, 2 Christian Goldhahn,1,3 Jonas Schubert, 2,4 Maximilian Johannes Hartl 5 and Munish Chanana 1,2,3 * 1. Department of Building Materials (IfB), ETH Zürich, 8093 Zürich, Switzerland 2. Department of Physical Chemistry II, University of Bayreuth, 95440 Bayreuth, Germany 3. Laboratory for Applied Wood Materials, EMPA Dübendorf, 8600 Dübendorf, Switzerland 4. Leibniz-Institut für Polymerforschung Dresden e.V. (IPFDD), 01069 Dresden, Germany 5. Department of Biopolymers, University of Bayreuth, 95440 Bayreuth, Germany
Abstract: In this work we show that different enzymes, such as horseradish peroxidase, glucose oxidase, laccase and catalase, can be directly immobilized onto plasmonic gold nanoparticles (NPs) and superparamagnetic iron oxide NPs simply via unspecific physical adsorption, yielding catalytically active and colloidally stable NP systems. The enzyme coating on the NP surface is highly robust and enzymatically active. The colloidal stability and the enzymatic performance
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(Vmax, Km) of the enzyme-coated NPs (Au@Enzyme) are strongly dependent on the pH of the dispersion and physicochemical properties of the respective enzyme. In particular, we observe that the colloidal stability of the enzyme-coated NPs does not necessarily lie in the pH range of the optimal catalytic performance of the enzymes (Au@HRP is unstable at pH 3 but kcat it the highest with 7.4 • 103 min-1). Understanding the relationship between the colloidal stability of the different enzyme-coated NPs at different pH values and the optimal catalytic performance of enzyme-coated NPs, will allow us to perform enzymatic reactions at the colloidal level, ensuring quasi-homogenous catalysis. We also demonstrate that the immobilization of different enzymes on different NP systems allows for designing enzymatic cascade reactions at an inter-colloidal level, with selective catalyst separation.
Keywords: Enzyme-coated nanoparticles, colloidal stability, protein adsorption, protein corona, enzyme immobilization, pH dependent enzymatic activity.
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Metal and metal oxide nanoparticles (NPs) were coated with various enzymes via physisorption, yielding catalytically active and colloidally stable enzyme-coated nanoparticles, with a robust enzyme coating. For example, HRP-coated Au NPs (purified) catalyze the oxidation of luminol, which is accompanied by the emission of light (chemiluminescence).
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Introduction: In the view of various biomedical and biotechnological applications of enzymes, the demands toward their stability, catalytic activity under non-optimal conditions, recovery and multiple reuse are high and urgent.1-4 An inevitable approach to meet these demands is their immobilization on solid supports. Parameters, such as the geometry of the support (e.g., flat surfaces, particle size and shape) as well as the surface properties (e.g. porosity, roughness, specific surface area/ pore size, surface chemistry, surface charge etc.) play an important role, making an adequate selection of the support crucial. In contrast to the commonly used stationary and non-stationary solid supports (heterogeneous catalysis), including porous and non-porous beads and particles, nanoparticles (NPs), in particular metal (Au, Ag) and metal oxide (e.g. Fe3O4) NPs represent a highly interesting class of supports for enzyme immobilization. Owed to their size related properties and advantages, such as high surface-to-volume ratios,5 diffusion coefficients close to that of molecules and plasmonic or magnetic properties, NPs allow for high immobilization efficiencies, quasi-homogeneous catalysis6-9 and facile plasmonic detection or magnetic separation, respectively. More importantly, by combining various enzyme-coated NPs (NP1@Enzyme1 + NP2@Enzyme2) the step towards (multi-) enzymatic cascade reactions at an inter-colloidal level becomes feasible. Co-immobilizing different enzymes on a single particle on the other hand, could provide a fine control over inter-enzyme distances, substrate channeling, thus allowing for fast multi-enzymatic cascade reactions at an intra-colloidal level.10-11 Furthermore, since the enzymes are immobilized on the (outer) surface of the NPs, the biocatalytic reactions are not subjected to diffusion and gradient problems (substrate, product, local pH), as it the case for porous supports.12
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However, for developing a NP-based functional and reusable (multi-) enzymatic system, the preservation of the colloidal stability of the NPs as well as the retention of the catalytic activity of the immobilized enzymes throughout all the involved steps and processes (i.e. enzyme immobilization on the NPs, application in biocatalysis, recovery and reuse) is of uttermost importance and represents a great challenge. For example, the multiple reaction and purification steps and changing reaction conditions (pH, ionic strength) can have a negative impact on the colloidal stability of the NPs (irreversible aggregation) as well as on the stability and catalytic activity of the enzymes (denaturation, loss of the secondary and/or tertiary structure.1, 12-16 Furthermore, enzyme immobilization requires, in particular for biocatalytical reactions, a strong enzyme-support attachment to avoid undesired release/detachment of the enzyme during its operation. Hence, among the various immobilization techniques12-13 (covalent, bioconjugation or physisorption), the prevalently applied immobilization techniques are mainly based on covalent binding of enzymes on to the nanoparticles,5, 7, 13-14, 17-18 using the strategy of multipoint covalent attachment via epoxide,16 glutaraldehyde19 or glyoxyl groups20 on the support side, ensuring permanent attachment and increased enzyme stability.13, 16 Although, physical adsorption of proteins on solid supports is perhaps the most simple and gentle technique for the enzyme immobilization, the risk of enzyme desorption is considered to be its main problem.12 In our recent work we have reported on physical adsorption of proteins on metal NPs (Au, Ag) of different sizes and shapes, yielding colloidally stable protein-coated metal NPs, with a robust layer (corona) of a defined protein.21-28 The protein adsorption/immobilization is highly robust, owed to multipoint attachment of the various functional groups available on the protein (e.g. thiol-, disulfide-, amino-, carboxylic, imidazol groups), since the protein would not detach from the metal NP surface, even after multifold purification steps. However, the impact of
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such a physisorption-based (multipoint) immobilization technique on the function of the protein, e.g. the catalytic activity of enzymes, has not been studied so far. The issues regarding the loss of the structure (denaturation, loss or distortion of tertiary (even secondary) structure) of enzyme upon their unspecific physical adsorption on to the metal NPs surface have also not been sufficiently resolved yet in the literature. Since aggregation of enzymes (e.g. at pH=pI) is one of main sources of their activity loss,13 the inter-relationship between the colloidal stability (agglomeration/aggregation) and the catalytic performance of the enzyme-immobilized NPs becomes an important subject, which has to be understood in order to develop colloidal enzymatic systems. Understanding the relationship between the NP identity, protein/enzyme identity and the environmental parameters (e.g. pH) is crucial to ensure the optimal catalytic performance of enzymes in an immobilized-state, but also to guarantee high colloidal stability of the NP@Enzyme systems, ensuring quasi-homogenous catalysis6-9 and enzymatic cascade reactions.29-30 In particular, pH is a crucial parameter, since both, the catalytic activity of enzymes, but also the colloidal stability of the protein/enzyme coated NPs are strongly pHdependent. Furthermore, pH-dependent reversible agglomeration can be used as a switch to toggle between quasi-homogeneous and heterogeneous catalysis by switching between colloidally stable and agglomerated NPs. Thus, in the present study, we firstly aim to investigate, if different enzymes retain their catalytic activity upon unspecific physical adsorption (physisorption) onto metal and metal oxide NPs. Secondly, we seek to understand the effect of pH on the correlation between the colloidal stability and the catalytic activity of the enzyme-coated NPs. And finally, we aim to develop an
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inter-colloidal (multi-) enzymatic cascade reaction system with facile catalyst recovery and reusability.
Figure 1: Enzymatic reactions of enzyme-coated NPs. A) Au@HRP NPs catalyzed oxidation of TMB to TMBDI in the presence of H2O2. The color of the dispersion turns brownish-yellow due to the color of the agglomerated NPs (brownish red) and of TMBDI (yellow). B) Same reaction as in (A) with HRP-coated Fe3O4-NPs. C) Au@Lac NPs catalyzed oxidation of TMB in the presence of O2. The color of the dispersion turns from purple to green. D) Au@GOx NPs catalyzed oxidation of β-D-glucose to gluconic acid, yielding H2O2, which used by the native
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HRP in the solution to oxidize TMB. E) Au@Cat NPs decomposes H2O2 to H2O and O2 (vigorous evolution of gas bubbles).
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Results and discussion: To demonstrate the effect of physisorption of enzymes on their catalytic activity, we coated citrate-stabilized gold NPs (Au@Citrate NPs, d = 16 ±1 nm, see Figure S1 in the supporting information (SI)) in a ligand-exchange process with four different enzymes (Figure 1). The enzymes used were: horseradish peroxidase (HRP), laccase (Lac), glucose oxidase (GOx) and catalase (Cat) (see Table 1 in SI). HRP was used as an exemplary enzyme to coat NPs of another surface chemistry, in this case Fe3O4-NPs, yielding Fe3O4@HRP NPs (Figure 1B). The enzymecoated NPs were purified from the excess enzyme and the free citrate molecules via five-fold centrifugation/redispersion steps or via magnetic separation in case of Fe3O4-NPs and were characterized via UV-Vis, DLS and TEM (Figure S1). The complete removal of unbound (free) enzymes from the enzyme-coated NP dispersions was certified as the supernatant of the fifth purification step did not show any protein absorbance peak (HRP 403 nm, GOx 280 nm) in UVVis spectrum. The immobilization yield was determined via UV-Vis spectroscopy and confirmed by theoretical calculations following the procedures reported elsewhere.31-32 Au NPs are particularly suited for such a study, owed to their plasmonic properties. The LSPR (localized surface plasmon resonance) of the NPs is highly sensitive to interparticle distances and serves as an indicator for the colloidal stability of the NPs (i.e. aggregation) during all procedures, including coating, purification and catalytic reaction steps. The Au NPs exhibited a slight LSPR redshift (3-7 nm) upon enzyme coating, due to the change in the refractive index around the NP surface, indicating direct immobilization of the enzymes on the NP surface and the replacement of the citrate, without inducing any NP aggregation (Figure S1), being in consistency with the Au@protein NPs systems. In the case of Fe3O4@citrate NPs, the enzyme coating was successful, too (see Figure S1). The final purified enzyme-coated Au- and
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Fe3O4-NPs exhibited high colloidal stability at pH values above the pI of the respective enzyme, e.g. at physiological pH of 7.4 (Figure S1), and agglomerate reversibly at pH =pH, inconsistency with similar protein-coated NPs.21-26 Remarkably, all the enzymes retained their catalytic activity when physisorbed onto the NPs (Figure 1). In order to demonstrate the catalytic action of the various NP@Enzyme systems, the chromogenic substrate tetramethylbenzidine (TMB) was employed for the Au@HRP, Fe3O4@HRP, Au@Lac and Au@GOx NPs. TMB is oxidized by the enzyme-coated NPs in the presence of H2O2 (NP@HRP, Figure 1A and B) or O2 (Au@Lac, Figure 1C), yielding a blue colored product (TMB•+) upon 1-electron oxidation and/or a yellow colored product (tetramethylbenzidine diimine (TMBDI)) upon a 2-electron oxidation (Figure S2). In the case of Au@GOx NPs (Figure 1D), β-D-glucose is oxidized in the presence of O2 to D-glucono-δlactone and equimolar amount of H2O2, which can be then used to oxidize TMB with the help of HRP, resulting in a typical colorimetric signal. In the case of the Au@Cat system, the vigorous evolution of gas bubbles (O2) in presence of H2O2 was observed (Figure 1E), demonstrating the catalytic activity of the Au@Cat NPs. To ensure that the catalytic performance of the enzyme-coated NPs is solely attributed to the immobilized enzymes, several control (blank) experiments were also performed (See Figure S3). In the case of HRP/TMB/H2O2 system, Au@Citrate (no protein) and Au@BSA NPs (no enzyme) were mixed with TMB and H2O2 respectively, and no reaction was observed. This result was also confirmed with an alternative reaction, namely the HRP-mediated oxidation of luminol in the presence of H2O2. HRP oxidizes luminol in the H2O2, yielding immediately a chemiluminescent signal. The reaction was performed with Au@Citrate, Au@BSA, Au@HRP (see Figure S4). Furthemore, to ensure the absence of any unbound (free) enzymes in the HRP-
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coated NP dispersions, the supernatant of the fifth purification step was also checked. The photoluminescent signal was only observed for Au@HRP system.
For Au@HRP NPs (Figure 1A), the pH of the NP dispersion was adjusted to pH 4.7, which is the optimal catalysis pH for HRP.33 At this pH, the Au@HRP NPs exhibit a pink-violet color with an LSPR peak around 558 nm, indicating agglomeration of the Au@HRP NPs, since the pH is close to the pI of HRP (pI = 6). Please note that this agglomeration is fully reversible (similar to other protein-coated NPs)21 and the Au@HRP NPs re-disperse completely by increasing the pH > pI, reaching the original LSPR maximum at 524 nm (Figure S1). Despite the agglomeration at pH 4.7, upon the addition of H2O2 and TMB and incubation time of 30 min, the color of the dispersion changes to brownish yellow due to the conversion of TMB to TMBDI. The final color of the dispersion is a combination of the yellow color of the diimine and violet color of the agglomerated Au@HRP NPs. A similar reaction was observed for Fe3O4@HRP NPs (Figure 1B). The Fe3O4@HRP NP dispersions are dark brown in color and exhibit superparamagnetic properties, making them highly useful for a facile magnetic separation and recovery of enzymecoated NPs. In order to maintain their colloidal stability, and to show their catalytic activity at physiological pH, the pH of the dispersion was adjusted to pH 7.4. To demonstrate the catalytic activity of the immobilized HRP on Fe3O4 NPs, H2O2 and TMB was added and after a short incubation time of approximately 1 minute, the Fe3O4@HRP NPs were removed with the help of a strong magnet. The solution color appeared to be yellow-orange proving the conversion of TMB. Au@Lac NPs are also able to oxidize TMB, consuming oxygen, which readily dissolves from the air into the dispersion. The pH of the Au@Lac NP dispersion was adjusted to pH 4.7 for the
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optimal catalytic conditions of Lac. The Au@Lac NPs at this pH exhibit deep purple color, with an LSPR peak at 580 nm, which shows NP agglomeration (Figure 1C). Immediately after the addition of TMB and gentle agitation, the dispersion turns green. The greenish color is indicative for a mixture of TMB•+ (blue) and TMBDI (yellow) along with the purple color of the NPs (Figure 1C). Just as well Au@GOx NPs exhibit catalytic activity, although the NPs are not colloidally stable at the pH of the enzyme´s catalytic pH optimum (pH 4.7). The Au@GOx NPs dispersion exhibits purple color with an LSPR peak at 585 nm (Figure 1D) and agglomerates of 834 nm in DLS. Upon addition of glucose, the Au@GOx NPs nevertheless catalyze the oxidation of glucose, producing 1 equivalent of H2O2 as a side-product. In the presence of HRP and TMB, the in-situ produced H2O2 molecule is used to subsequently oxidize TMB, giving TMB•+ product that is blue in color (Figure 1D). The enzymatic reaction for the Au@Cat NPs was performed at pH 7.4, at which they are highly stable and exhibit a deep red color with a LSPR peak at 527 nm. Catalase degrades H2O2 vigorously into H2O and O2 gas, which can be easily seen as bubbles by the bare eye (Figure 1E). As a control, Au@BSA NPs were employed (Figure S5 Au@BSA+H2O2). In summary, all used enzymes retained their catalytic activity upon adsorption onto NPs. It is worth to note that most of the enzyme-coated NPs shown in Figure 1 are not colloidally stable at the optimal pH of their enzymatic reaction (see left cuvettes), which can be seen by the color of the dispersions.21 Nevertheless, Figure 1 clearly shows that the enzyme-coated NPs are all enzymatically active over a broad range of pH, but also in agglomerated and individually dispersed state.
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The intrinsic complexity of a protein-coated nanoparticle system (NP@Protein), in particular owed to the diversity of proteins and their abundance in the environment, makes it challenging to predict the final physicochemical properties of the NP@Protein system, but also the nature of the protein-coating on a NP.34 The physicochemical parameters of the NPs (NP identity, i.e. the surface chemistry, size and morphology), the molecular and physicochemical properties of the protein (protein identity, i.e. primary, secondary and tertiary structure, isoelectric point (pI), distribution of charge and hydrophilic/hydrophobic domains) and the environmental conditions (pH, ionic strength and temperature) are strongly interrelated with each other (during the adsorption process,21 but also in the immobilized state34). Therefore, depending on these parameters, the protein-coated NPs exhibited different stability profiles under different environmental conditions.21 In general, all protein-coated NPs exhibit a strong pH-dependent colloidal stability profile21-25 featuring an isoelectric point (NP pI), mostly shifted towards acidic pH from the original pI of the protein (protein pI).21 As a consequence, the Au@Protein NPs are highly stable at pH values below and above their pI (NP pI) and agglomerate (reversibly) at pH values close to their pI. In order to make use of such enzyme-coated NPs in sophisticated catalysis systems, it becomes necessary to investigate the optimal reaction conditions (like pH) for the adsorbed enzymes, which may be significantly different to the optimum of unbound enzymes. Furthermore, the colloidal stability of the NPs is believed to have an influence on the overall catalytic performance of the enzyme-coated NPs. Depending on the environmental conditions, such NPs can stay individually dispersed (quasi-homogeneous system) or agglomerate and even sediment (heterogenous system), which surely will have an effect on the reaction rates. Furthermore, upon
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aggregation (e.g. at the pI)35 the active centers of the enzymes can become unavailable, leading to the loss of activity of the enzyme, which is also the case for free enzymes.13
In order to assess colloidal stability profiles of the various Au@Enzyme NPs in dependence of the environmental pH, pH-dependent UV-Vis and DLS measurements in the relevant pH range of optimal catalytic activity were performed (Figure 2). Figure 2 shows that all the Au@Enzyme NPs are highly stable above pH 7, which is significantly above the pI of the enzymes (see Table S1). In the pH range around the pI of the enzymes, the NPs become unstable and start agglomerating, with strongest agglomeration at pH ≈ pI, suggesting that most of enzymes used here have their pI between pH 4 and 6. The Au@Enzyme NPs agglomerate accordingly around pH 4-5. These results on the pH-dependent colloidal stability of Au@Enzyme NPs are highly consistence with our previous studies on gold NPs, coated with proteins of similar pI (i.e. pI 4-6), and their physicochemical properties in regard to the pH of the dispersions.21-25 Accordingly, at pH values far below and above the pI (pH>pI, pH
