Rational Nanoconjugation Improves Biocatalytic Performance of

Article pubs.acs.org/Langmuir

Rational Nanoconjugation Improves Biocatalytic Performance of Enzymes: Aldol Addition Catalyzed by Immobilized Rhamnulose-1Phosphate Aldolase Inés Ardao,†,⊥,# Joan Comenge,‡,§,⊥ M. Dolors Benaiges,† Gregorio Á lvaro,*,† and Víctor F. Puntes*,‡,∥ †

Departament d’Enginyeria Química, Universitat Autònoma de Barcelona, Unitat de Biocatàlisi Aplicada associada al IQAC (UAB-CSIC), Barcelona, Spain ‡ Catalan Institute of Nanotechnology (ICN), Centre d’Investigació en Nanociència i Nanotecnologia (CIN2), and Universitat Autònoma de Barcelona (UAB), Barcelona, Spain § International Iberian Nanotechnology Laboratory (INL), Braga, Portugal ∥ Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain S Supporting Information *

ABSTRACT: Gold nanoparticles (AuNPs) are attractive materials for the immobilization of enzymes due to several advantages such as high enzyme loading, absence of internal diffusion limitations, and Brownian motion in solution, compared to the conventional immobilization onto porous macroscopic supports. The affinity of AuNPs to different groups present at the protein surface enables direct enzyme binding to the nanoparticle without the need of any coupling agent. Enzyme activity and stability appear to be improved when the biocatalyst is immobilized onto AuNPs. Rhamnulose-1-phosphate aldolase (RhuA) was selected as model enzyme for the immobilization onto AuNPs. The enzyme loading was characterized by four different techniques: surface plasmon resonance (SPR) shift and intensity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). AuNPs-RhuA complexes were further applied as biocatalyst of the aldol addition reaction between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal during two reaction cycles. In these conditions, an improved reaction yield and selectivity, together with a fourfold activity enhancement were observed, as compared to soluble RhuA.

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INTRODUCTION Inorganic nanoparticles (NPs) have become of great interest as scaffolds for protein immobilization in different fields such as biocatalysis,1 biosensing,2 and biomedicine.3 Proteins are being immobilized either by physical adsorption to the NPs surface4 or by covalent bonding to previously functionalized nanoparticles.5 In particular, enzyme immobilization onto NPs presents several advantages compared to the use of conventional methods such as immobilization onto porous macroscopic supports:6 high enzyme loadings can be obtained due to the NPs maximized surface per unit mass, internal diffusion limitations are avoided, and external agitation might be spared since NPs undergo Brownian dispersion efficiently.7,8 Of special interest is the enhancement of the enzymatic activity and stability,9,10 and change in substrate specificity11 that is observed when enzymes are immobilized onto NPs, in comparison to free enzyme or onto classic immobilization supports. However, despite the interest and efforts, there are still several questions to be solved, for example, the control of loading and the stability of the protein, before taking full advantage of NP-protein complexes. A key issue in enzyme immobilization onto AuNPs is to find compatible environments for coupling enzyme performance to © 2012 American Chemical Society

the colloidal stability of nanoparticles. Proteins usually need high ionic strengths and pH close to neutrality in order to exhibit high enzymatic activity and stability,12,13 whereas NPs tend to aggregate in these conditions, thus leading to the loss of their special properties.14 Fortunately, after the formation of the AuNPs-enzyme complex, the protein shell is known to favor the colloidal stability of NPs15 and the NP to confer extra-stability to the protein.16 Nevertheless, the structure, stability and reactivity of the enzyme may vary during conjugation and therefore compromise its biocatalytic performance.17 Structural modifications of enzymes are translated in modifications of its reactivity.18 Additionally, special care should be taken during the process of conjugation to prevent a denaturation and oversaturation of protein at the surface leading to the formation of multilayers.19 In this undesired case, diffusion limitations of the reactants to the inner layers of protein or even the inactivation of the enzymes would occur. Rhamnulose-1-phosphate aldolase from E. coli (RhuA, EC 4.1.2.17, isoelectric point 5.5) has been chosen as a model Received: January 27, 2012 Revised: March 19, 2012 Published: March 19, 2012 6461

dx.doi.org/10.1021/la3003993 | Langmuir 2012, 28, 6461−6467

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suspension (1 mL). The mixture was measured by UV−vis spectroscopy after 30 min to determine whether the conjugates stability was maintained or the formation of aggregates was induced. Desorption Study of AuNPs-RhuA Conjugates. AuNPs-RhuA from the centrifugation of immobilization mixture (10 mL) were suspended in reaction medium 50 mM Tris−HCl, 150 mM KCl, and 20% v/v dimethylformamide (DMF), pH 7.0 (0.4 mL total volume). The mixture was kept at 4 °C without agitation during 28 h. A control of soluble RhuA stability was performed by incubating a solution of RhuA (initial activity of 0.9 AU·mL−1) under the same conditions. At different times, homogeneous samples of suspension were withdrawn and the RhuA activity was tested. Supernatant activity was determined after centrifugation (Heraeus Pico-21, 18 800g, 3 min, 4 °C) of a suspension sample (50 μL). The enzymatic activity (AU/mL−1) was measured using a coupled enzymatic assay according to literature.28 One activity unit of RhuA was defined as the amount of enzyme required to convert 1 μmol of rhamnulose 1-phosphate in DHAP per minute at 25 °C. Aldol Addition Catalyzed by AuNPs-RhuA. AuNPs-RhuA from the centrifugation (Beckman J2-21, 11 700g, 10 min, 4 °C) of immobilization mixture (25 mL) were added to the reaction medium (1 mL total volume). The final concentration was 30 mM DHAPLi2, 30 mM (S)-Cbz-alaninal, 50 mM Tris−HCl; 150 mM KCl; 20% v/v DMF, pH 7.0. The reaction medium was prepared by dissolving the (S)-Cbz-alaninal in DMF and DHAPLi2 in the aqueous components of the reaction medium and subsequent mixing, due to the different hydrophobicity of these reactants. The reaction suspension was incubated during 20 h at 4 °C without agitation. Homogeneous samples of suspension (50 μL) were withdrawn at different times, diluted 1:4 with methanol to stop the enzymatic reaction, and centrifuged (Heraeus Pico-21, 18 800g, 2 min, 4 °C) to remove the precipitated biocatalyst. The concentrations of DHAP, (S)-Cbzalaninal, and product in the supernatant were determined. DHAP concentration was measured in a spectrophotometer (Cary 50 UV− vis, Varian) by reaction with reduced nicotine adenine dinucleotide (NADH) catalyzed by rabbit muscle glycerol 3-phosphate dehydrogenase (GDH).23 (S)-Cbz-alaninal and the synthesis product were measured on an HPLC system (Dionex Ultimate 3000) fitted with a X-Bridge C18 5 μm 4.6 × 250 mm column (Waters) with chromatographic conditions previously reported.29 In the case of the aldol addition catalyzed with soluble enzyme, the amount of precipitated RhuA that corresponds to an equivalent number of activity units was dissolved in the reaction medium after centrifugation (Heraeus Pico-21, 11 700g, 3 min, 4 °C) of the storage suspension and treated in the same way. AuNPs-RhuA from the reaction mixture after 19 h of operation were employed as biocatalysts in a second cycle of reaction in order to evaluate their reusability. The first-cycle mixture was centrifuged (Heraeus Pico-21, 11 700g, 3 min, 4 °C) and fresh reaction medium was added to the AuNPs-RhuA (0.65 mL total volume). The final concentration was 30 mM DHAPLi2, 30 mM (S)-Cbz-alaninal, 50 mM Tris−HCl, 150 mM KCl, and 20% v/v DMF, pH 7.0. Samples were withdrawn and treated as stated above. The initial activity of both cycles was determined in a parallel experiment, in order to overcome the interference of the presence of DHAP in the enzymatic activity test.23 In the first cycle, an aliquot of immobilization mixture equal to the one used in the reaction was centrifuged at the same conditions and suspended in reaction medium to the same final concentration but in the absence of reactants. The enzymatic activity of this suspension was determined and taken as the initial activity of the aldol addition reaction. This mixture received the same treatment as the reaction mixture, including the centrifugation after 19 h of operation at the same conditions. The AuNPs-RhuA were subsequently resuspended in reaction medium with no reactants to the final conditions of the second cycle of reaction. The enzymatic activity of this suspension was measured and considered as the initial activity of the second cycle of reaction.

enzyme for protein immobilization in AuNPs. This enzyme belongs to the family of lyases, specifically to the group of dihydroxyacetone phosphate (DHAP)-dependent aldolases, which cleave carbon−carbon bonds. In nature, RhuA catalyzes the reversible cleavage of L-rhamnulose-1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. In the synthetic direction, RhuA is very specific for DHAP as electron-donor but accepts a wide range of aldehydes, which has been exploited for synthetic applications.20,21 Specifically, RhuA catalyzes the stereoselective synthetic reaction between DHAP and (S)-Cbz-alaninal to render a precursor of iminocyclitols, a class of compounds with antiviral, anticancer and antidiabetic activities.22,23 In this study, the characterization of the different conjugation states provided strategies to differentiate between a fully covered monolayer, a partial layer or a multilayer of proteins on top of the NPs. Therefore, optimized conditions to achieve a complete coverage of the surface without compromising the stability of both the nanoparticles and the enzymes were determined. The biocatalytic performance of the AuNPs-RhuA conjugates was assessed using two different reactions: the synthetic aldol addition between DHAP and (S)-Cbz-alaninal (expressed as reaction rate, mM·min−1) and the natural reaction of L-rhamnulose-1-phosphate cleavage (expressed as activity units per mL, AU·mL−1) and compared to the results obtained with the equivalent of free enzyme. A fourfold enhancement of the enzymatic activity and an improvement in selectivity is reported here. This enhancement is maintained in subsequent reaction cycles.

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MATERIALS AND METHODS

Reagents. Gold(III) chloride trihydrate and sodium citrate were purchased from Sigma Aldrich (St. Louis, MO). RhuA from E. coli (EC 4.1.2.17) was produced and purified as a recombinant enzyme according to a previously published protocol24 and stored precipitated in ammonium sulfate solution at 4 °C. Dihydroxyacetone phosphate dilitium salt (DHAPLi2) was supplied from Fluka (Buchs, Switzerland) and (S)-Cbz-alaninal was purchased from Sunshine Chemlab (Downingtown, PA). The dicyclohexylammonium salt of rhamnulose-1phosphate was synthesized according to reported procedure.25 Other reagents were of analytical grade. Synthesis of Gold Nanoparticles. AuNPs (47 nm) were synthesized following a seeding growth mechanism based on the standard method of gold salt reduction by citrate described in the literature.26 Specifically, three consecutive steps consisting of 1 mL injection of 25 mM HAuCl4 to previously synthesized AuNPs were done, achieving a AuNPs final concentration of 4.5 × 1010 NP·mL−1. UV−vis spectrum of the AuNPs was determined at wavelengths from 800 to 300 nm (UV-2401 PC, Shimadzu). Particle size was measured by transmission electron microscopy (TEM) imaging (Jeol 1010) and the hydrodynamic diameter by dynamic light scattering (DLS) (Zetasizer nano ZS 90, Malvern Instruments). Loading of RhuA onto AuNPs. Different amounts of a RhuA solution of 0.5 mg of protein per mL (determined by Bradford assay27) in 2.2 mM sodium citrate pH 6.7 were added to 1 mL aliquots of 47 nm AuNPs suspension in 2.2 mM sodium citrate (4.5 × 1010 NP·mL−1) to a final volume of 1050 μL in all experiments. Final RhuA concentrations went from 0 to 189 nM. The mixture was incubated during 30 min under soft stirring conditions at room temperature. The resulting conjugates were measured by UV−vis spectroscopy and DLS in order to determine the peak position and intensity of the SPR as well as estimate the size of AuNPs-RhuA. Flocculation Test. AuNPs-RhuA conjugates with different coverage were prepared as explained above. The conjugation was kept running during 2 h to ensure that the equilibrium was reached. Afterward, 1 M NaCl (50 μL) were added to this AuNPs-RhuA 6462


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RESULTS AND DISCUSSION Characterization of the Loading of RhuA onto AuNPs. a. Monolayer Formation and AuNP-RhuA Conjugates Characterization. Gold nanoparticles (AuNPs) have been previously used in protein immobilization due to their interesting size and controlled physical and chemical properties compared to other inorganic nanoparticles.30 Different groups present in proteins such as thiols, carboxylic acids, and amines allow their adsorption on Au surfaces readily without any additional modification.1 The attachment of RhuA onto AuNPs has been studied by adding different concentrations of RhuA from 1 to 180 nM to a suspension of 47 nm diameter AuNPs (Figure S1, Supporting Information) at fixed concentration (0.1 nM, 4.5 × 1010 AuNPs·mL−1). Large AuNPs were chosen in order to accommodate a big enough number of enzyme molecules per AuNP and because they host the protein without denaturation due to the curvature radii. Three different readings were used to characterize the loading of enzyme: the intensity and the red-shift of the AuNPs surface plasmon resonance (SPR) peak in the UV-spectrum (caused by the interaction with the surface electrons of the metallic NP),31 and the conjugate size measured by dynamic light scattering (DLS, a technique sensitive to the hydrodynamic size of species in solution) (Figure 1). At the employed NP/RhuA relative

together with an increase of the peak intensity going from 1.05 when no RhuA was added, to 1.22 once the saturation plateau was reached. This increase in absorbance was not due to any NP concentration effect, since the AuNPs concentration was kept constant through all the experiments. In parallel, the hydrodynamic diameter of the conjugates was measured by DLS and a steady increase was observed as enzyme was added, also reaching a plateau at the same concentration (70 nM). The observed increase in the hydrodynamic diameter is related to the progressive formation of a more compact protein shell (the measured hydrodynamic diameter represents the diameter that a sphere with the same translational diffusion speed than the conjugates would have).33 The hydrodynamic diameter of naked AuNPs was 54 nm while the fully loaded AuNPs-RhuA presented a value of 70 nm. This 16 nm increase is in agreement to a protein monolayer model taking into account that RhuA can be considered as a 7 × 7 × 5 nm3 prism.34 It should be noted that size distribution was not modified after adsorption of RhuA (Figure S2, Supporting Information). Similar profiles have been obtained with the different techniques employed to follow the loading of RhuA onto AuNPs. In all cases a rapid increase of the signal is observed between 0 and 70 nM, followed by a plateau at higher concentrations. This plateau indicates that AuNPs were completely loaded with enzyme since subsequent addition of enzyme had no effect in the signal of the three different techniques. This can be correlated to the Langmuir adsorption isotherms with the following considerations. The model assumes that adsorption at one site does not affect the energy of adsorption at neighboring sites. However, adsorption of proteins is affected by the presence of other proteins. This accounts for the moderate deviation from the Langmuir Isotherm (Figure 1). In this case, higher RhuA concentrations are necessary to reach the same capping grade than the one expected by Langmuir’s model.31 The RhuA saturation point on the AuNPs surfaces (point D in Figure 1) can be established at 70 nM, which would correspond to 928 molecules of RhuA per AuNPs (if all RhuA was absorbed onto NPs). This discrepant value is 7-times higher than the one corresponding to a theoretical monolayer, which is estimated to be 128 molecules of RhuA per AuNP. This theoretical value is calculated by taking into account the diameter of naked NP measured by TEM (46.5 ± 4.7 nm), the theoretical radius of RhuA (estimated to be 3.9 nm),34 and a packing factor of 90%, which corresponds to the close-packing density of spheres in a surface (presuming enzymes are spherical). This difference represents the excess of enzyme needed to deposit a full monolayer in 30 min at the employed concentrations and reflects the equilibrium between proteins in solution and proteins on the surface of the NP. It is expected that the exchange rate will slow down with incubation time due to the hardening of the protein layer.32 This excess value agrees with other works in literature where 5- to 10-fold excess are needed to achieve a protein monolayer coverage.7,35 Despite there is one superficial cysteine (which contains a thiol group) per subunit of RhuA available to bind AuNPs via covalent-like SH−Au bonding,36 we believe that the interactions of RhuA with AuNPs are more likely to be mainly via multiple weak interactions, such as the ones formed with amines or carboxylic acids from basic or acid residues present in a great number in the enzyme surface.37 This would explain the excess needed to achieve the full monolayer as well as the partial desorption after resuspension in fresh media (vide infra).

Figure 1. Loading of AuNPs with RhuA. Correlation of the SPR peak wavelength (hollow squares), SPR intensity (blue triangles), and hydrodynamic diameter measured by DLS (green squares) with the initial RhuA concentration added. A system that accomplishes the Langmuir approach (e.g 11-mercaptoundecanoic acid) was simulated in order to compare with the behavior of enzymes (dashed line). (Inset) Schematic diagram of the variation of the DLS-measured hydrodynamic diameter in function of the compactness of the protein shell. It is shown the main states of the AuNPs, namely, naked AuNPs (A), nonstable against flocculation test AuNPs (B), stable against flocculation (C), and saturated AuNPs-RhuA (D).

concentrations, proteins reach the AuNPs surface almost instantaneously after mixture and readily conjugate.32 In all cases, a rapid evolution was observed at very short times (seconds to minutes) and no further changes in the UVspectrum of the mixture were observed after 30 min (and even 24 and 48 h later), which confirmed that this time was enough to reach the final state of the conjugate. A peak shift from 527.8 nm (naked, citrate capped AuNPs) up to saturation at 533.3 nm for RhuA concentrations higher than 70 nM was observed, 6463


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nonconjugated state, distances ≥ 10 nm are observed between the conjugated NPs. b. Colloidal Stability and Desorption of Enzyme. In order to further study the structure of the conjugates, we analyzed the colloidal stability of the formed species. The flocculation test is often used in literature to determine the point at which the nanoparticles are sufficiently loaded to prevent their aggregation.10,37 This method consists of the addition of a high NaCl concentration to a suspension of enzyme−AuNPs conjugates. When the surface of the AuNPs is not covered enough by enzyme molecules, the salt ions compact the Stern layer, which is related to the electrostatic stability of the conjugates, and therefore the nanoparticle surfaces are able to interact leading to aggregation.38 Once the surface is coated with a certain number of molecules, the particle is sterically protected against aggregation. This happens before full coverage is reached. Thus, the stability point is reached at lower concentration than the saturation point, which is in agreement with previously reported results.37 In the case of RhuA immobilization onto AuNPs, the concentration of enzyme that avoids AuNPs aggregation against 50 mM NaCl was 16 nM (Figure 4), which is nearly 4 times lower than the

Once the surface of the AuNPs is fully covered by enzyme, an excess of RhuA in solution could induce the formation of enzyme multilayers.19 However, no further increase in the particle hydrodynamic diameter after saturation was observed by the DLS measurements performed, indicating no signs of enzyme multilayer formation on top of the NPs. In order to verify the capability of this technique to detect RhuA multilayers onto AuNPs, we induced the formation of multilayers by incubating the AuNPs-RhuA conjugates with an excess of enzyme at higher temperature to promote denaturation and force protein aggregation. A further increase of the DLS-measured hydrodynamic diameter was clearly observed (Figure 2), confirming that this technique is able to

Figure 2. Multilayer formation induced by denaturation of RhuA. The changes in size due to denaturation were measured by DLS. The increase of temperature led to conformational changes of the protein and consequently to the formation of a complex network between AuNPs and RhuA that finally precipitated. In comparison with free RhuA, it is showed to demonstrate that the formation of multilayers was induced by denaturation of enzyme.

distinguish between monolayer and multilayer coverage. Therefore, the formation of multilayers during RhuA immobilization onto AuNPs is discarded. Transmission electron microscopy (TEM) imaging after negative staining (Figure 3) confirms the binding of protein on

Figure 4. Flocculation test performed at increasing concentration of RhuA. 50 μL of 1 M NaCl was added to 1 mL of 47 nm AuNPs (4.5 × 1010 AuNP/mL) containing concentrations of RhuA in the range of 6.5−65 nM.

concentration corresponding to the saturation point previously determined (70 nM). When AuNPs-RhuA are resuspended in fresh medium, desorption of enzyme is expected since there is an equilibrium between adsorbed and nonadsorbed protein on the NP, specially in the case of proteins that are not strongly bonded.39 In order to quantify this desorption and evaluate whether the colloidal stability is compromised in a high ionic strength media, the conjugates were centrifuged and resuspended in reaction medium (containing 150 mM KCl, 50 mM Tris-HCl among others). The desorption of the enzyme in this media was calculated by measuring the activity of the system before and after removing the nanoparticles by centrifugation. After 2.7 h of incubation and further centrifugation, 38% of the total activity initially added was detected on the supernatant which clearly indicates that a partial desorption of enzyme is produced. Despite enzyme desorption, no aggregation occurred. This is in agreement with the flocculation test, where lower loadings of protein than the required to achieve a full monolayer are able to maintain colloidal stability in high ionic strength media. Interestingly, after the desorption event

Figure 3. TEM images of gold nanoparticles before (A) and after (B) conjugation to RhuA. Sample B was negative stained with 1% uranyl acetate previously to TEM imaging. RhuA excess was not removed to achieve better contrast. Scale bar is 100 nm in both cases.

the AuNPs surface. In these experiments, 1% uranyl acetate solution is soaked in the TEM sample. This agent stains the surface of the proteins, thus inducing a gray background in the TEM images when, as in our case, excess protein has not been removed before imaging. Wherever proteins are packed, the staining compounds cannot penetrate, resulting in a white area, as the hallo observed in the TEM image of the conjugated NPs. Note also that while contact between NPs is observed in the 6464


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RhuA is a very high efficient biocatalyst with a high turnover number of 6 × 106 s−1 in free form,24 defined as the maximum molecules of substrate converted into product per catalytic site of the enzyme and unit of time when the enzyme is saturated with substrate (5−10 times the KM). In the case of immobilized enzyme, 1 × 106 molecules of substrate were converted per catalytic site and second, even though enzyme is not saturated with substrate (initial DHAP concentration (30 mM) is 1 order of magnitude less than the corresponding KM for DHAP of soluble RhuA (174 mM)24). Therefore, an even higher turnover number for AuNPs-RhuA is expected under saturation conditions, confirming that the high catalytic efficiency of RhuA was preserved after immobilization onto AuNPs. It is well-known that the affinity of the enzyme for its substrates can be altered after their immobilization. Likely, for every enzyme and substrate, different changes in substrate affinity after immobilization and subsequent changes in the reaction rate may take place.4,10,16,4,43 In the case of RhuA immobilization onto AuNPs, the substrates of the natural reaction and the aldol addition are different in both chemical structure and size, and steric hindrance may take place. Additionally, during aldol addition, a ternary complex RhuA− DHAP−(S)-Cbz-alaninal needs to be formed to render the product, and this ternary complex is larger in size than the binary complex RhuA−Rhamnulose-1-phosphate needed in the natural reaction. Therefore, small conformational and orientational changes in the protein structure, induced by the immobilization, are more likely to have a greater effect in the binding of the most hindered complex, namely, the ternary complex formed in the aldol addition. This could lead to a higher enhancement of this reaction rate compared to the natural reaction. The possibility of reusing the AuNPs-RhuA in subsequent reaction cycles was also evaluated. The conjugates were softly centrifuged once the reactants of the first cycle of reaction were depleted. The centrifugation speed and time were carefully adjusted in order to avoid the irreversible aggregation of the particles. The AuNPs-RhuA were resuspended again in fresh medium for a second reaction cycle at the same initial reactant concentrations as in the first one. The initial reaction rate for the aldol addition in the second cycle (2.4 mM·h−1) was again 4-fold higher in comparison with the biocatalysis of free enzyme at the same conditions (0.56 mM·h−1). Thus, in addition to an increased activity, the recycling of the catalyst is also favored by the presence of the AuNPs.

the activity of the conjugates exhibited and almost constant value along 25 h (estimated half-life time of 50 days in comparison with 2.8 days of the soluble enzyme24). This fact means that no more desorption is produced after reaching a new equilibrium. On the other hand, a control solution of free enzyme lost 25% of the initial activity after the same incubation time due to enzyme deactivation. This shows that the attachment of RhuA to the AuNPs stabilizes the protein structure and subsequently reduces enzyme deactivation, as observed in related systems.40,41 Aldol Addition Catalyzed by AuNPs-RhuA. Finally, RhuA immobilized onto gold nanoparticles was used as biocatalyst in the aldol addition between DHAP and (S)-Cbzalaninal (reaction a, Figure S3, Supporting Information) and the natural reaction (cleavage of rhamnulose-1-phosphate) (reaction b, Figure S3, Supporting Information). The AuNPsRhuA activity was compared to the activity of the free enzyme under the same conditions and enzyme loading (Figure 5).

Figure 5. Evolution of the concentration of dihydroxyacetone phosphate (DHAP), (S)-Cbz-alaninal, and product in the biocatalysis with AuNPs-RhuA and free RhuA at 4 °C in medium 50 mM TrisHCl, 150 mM KCl, 20% v/v DMF, pH 7.0.

Here it is important to note the differences observed in the evaluation of the activity using different substrates. When using the natural substrate of the enzyme (L-rhamnulose-1phosphate), no increase in the activity was observed whereas a nearly 4-fold enhancement was observed with the substrates of the synthetic reaction (DHAP and (S)-Cbz-alaninal). The AuNPs-RhuA successfully catalyzed the aldol reaction with an initial reaction rate of 4.5 mM·h−1, whereas the reaction rate decreased to 1.1 mM·h−1 in the case of free enzyme. The selectivity of the reaction is limited by the degradation of DHAP catalyzed by the same enzyme, which occurs simultaneously to the aldol addition as an unwanted secondary reaction42 (reaction c, Figure S3, Supporting Information). Defining selectivity as the relation between the concentration of product and the total consume of the limiting reactant, we calculated it to be 73%. These values are slightly higher than those obtained with free RhuA at the same reaction conditions (67%) showing also improved selectivity. An improved final yield of 66%, defined as the ratio between the concentration of product and the initial concentration of the limiting reactant, compared to 59% in the case of free RhuA was also observed.

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CONCLUSIONS In summary, we have reported here different techniques to follow the conjugation of enzymes to AuNPs. Discerning between the formation of monolayers or multilayers as well as between the stability and the saturation points are of special interest in order to properly design and avoid misinterpretations in the characterization of these conjugates as biocatalysts. We also showed the relevance of studying the desorption of protein when it is adsorbed on the nanoparticle surface via noncovalent bonds, which is the case of most works regarding protein immobilization on nanoparticles found in the literature. More sophisticated approaches, like covalent bonding by EDC coupling could be undertaken, but it would render the technology unviable. Here, by simple conjugation, the overall reactivity, stability, selectivity, and yield have been improved and AuNPs conjugates are easily recovered for their next use without losses in reactivity. Several factors may influence the 6465


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observed enhancement of the activity. The presence of the AuNPs themselves, well-known catalysts that are able to increase electron transfer rates in redox reactions by acting as an electron donor/acceptor,8,44 cannot be discarded. However, there is some evidence that this enhancement is mainly due to the induction of small conformational changes that, without compromising enzyme stability, either facilitate the entrance of the reactants to the active site or stabilize the reaction intermediate,9,10,31 thus improving biocatalysis.

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ASSOCIATED CONTENT

S Supporting Information *

TEM characterization of AuNPs, scheme with the reactions catalyzed by RhuA, and DLS of AuNPs before and after RhuA conjugation. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

́ *(G.A.) Mailing address: Departament d’Enginyeria Quimica, Universitat Autònoma de Barcelona, Unitat de Biocatàlisi Aplicada associada al IQAC (UAB-CSIC), E- 08193 Bellaterra (Barcelona), Spain. E-mail: [email protected]. Telephone: +34 935812791. (V.F.P.) Mailing address: Catalan Institute of Nanotechnology (ICN), E- 08193 Bellaterra (Barcelona), Spain. E-mail: [email protected]. Telephone: +34 935868013. Present Address #

Institute of Bioprocess and Biosystems Engineering, Technische Universität Hamburg-Harburg. Hamburg, Germany. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Dr. Cecilia López for her critical reading of the manuscript. Inés Ardao acknowledges a predoctoral grant from DURSI (Generalitat de Catalunya) and European Social Funds. This work was supported by the Spanish MICINN, Project Numbers CTQ2008-00578, MAT 2009-147434-CO2-01, and CSD 2006-00012, research group 2009SGR281, 2009 SGR 776, and Biochemical Engineering Unit of the Reference Network in Biotechnology (XRB), Generalitat de Catalunya.

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REFERENCES

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