Inorganic Interfaces for

Oct 8, 2013 - ... degree in chemistry from the University of Peradeniya, Sri Lanka, in 2006. ... his Ph.D. from the Indian Institute of Technology Kan...
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Invited Feature Article pubs.acs.org/Langmuir

Nanobio Interfaces: Charge Control of Enzyme/Inorganic Interfaces for Advanced Biocatalysis Inoka K. Deshapriya† and Challa V. Kumar*,‡ †

Department of Chemistry and ‡Department of Molecular and Cell Biology, Institute of Material Science, 55 North Eagleville Road, Storrs, Connecticut 06226, United States ABSTRACT: Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications. and cyclization.14 Enzymes are the ultimate sustainable and renewable green catalysts derived from earth-abundant, lowcost elements from biological feed stocks by production methods that are also benign, renewable, and sustainable. Therefore, enzymes are versatile, promising candidates as industrial catalysts for the production of fine chemicals, pharmaceutical intermediates, and drugs as well as in food processing. The use of enzymes outside the biological environment, however, has several challenging limitations that include but are not limited to (1) the high cost of large-scale production, separation, and purification of enzymes; (2) their susceptibility to deactivation under nonambient conditions, organic solvents, and high ionic strengths; (3) their often very high specificity for a given substrate and therefore ineffectiveness as even closely related substrate analogues; and (4) the difficulty in recovering them from the reaction media after a catalytic reaction.15,16 Therefore, lowering their cost, enhancing their stability, broadening their specificity, and preventing their deactivation by organic solvents are worthy, beneficial, and challenging goals.

1. INTRODUCTION Enzymes bound to nanomaterials are being used extensively in advanced biocatalysis,1−3 biomaterial,4 and biomedical applications.5,6 This feature article describes our recent investigations of enzyme binding to inorganic nanomaterials and our current understanding of how to manipulate the nanobio interface in a predictable manner. The study of these interfaces is important to our understanding of fundamental biological processes such as enzyme binding to bone and teeth, binding of the viral particles to host−cell surfaces prior to infection, cell adhesion to solid surfaces, and cell migration. These activities can contribute to the fundamental understanding of nanobio interfaces and aid in the development of rational approaches to controlling these interfaces for practical applications or to engineering advanced biomaterials with predictable properties. Most enzymes are proteins, and they can be considered to be complex, large, well-defined, chiral organic molecules that often carry metal ions and water molecules to maintain their enzymatic activities and/or structure.7 Our studies mainly focused on enzymes because of their remarkable role as highly specific catalysts in living systems. They catalyze almost all chemical reactions in the biological world, under ordinary conditions of aqueous solutions, ambient pressures, and physiological temperatures.8 These reactions include hydrolysis,9 condensation,10 redox,11 isomerization,12 substitution,13 © 2013 American Chemical Society

Received: August 16, 2013 Revised: October 5, 2013 Published: October 8, 2013 14001

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Scheme 1. Enzyme Intercalationa

Approaches to control enzyme binding to α-ZrP. Exfoliation with tetrabutyl ammonium hydroxide (TBAOH) followed by (A) direct binding of positively charged enzymes, (B) metal ion-mediated binding of anionic enzymes, (C) Hb-mediated binding of DNA/RNA, (D) cationization of enzymes to promote their affinities, and (E) protein glues for the binding of enzyme−polymer conjugates. The % retention of hemoglobin peroxidase-like activity is shown in the parentheses for each approach.

a

then bound enzyme would be more stable than the corresponding pristine enzyme. Stabilizing the native state and destabilizing the denatured state (SNSDDS) by the nanobio interface therefore, is a powerful strategy. Because the equilibrium constant for enzyme denaturation (Kd) is exponentially related to ΔGd, a small improvement in ΔGd will provide significant gains in stability. Because the enthalpy change (ΔHd) is positive for enzyme denaturation, one obvious approach to increasing ΔGd and stabilizing enzymes is to decrease ΔSd. Our unique strategy to improve the enzyme stability has been to lower ΔSd by lowering the entropy of the denatured state of the bound enzyme. The entropy of any molecule depends on its conformational freedom (S = k ln Ω, where k is the Boltzmann constant and Ω is the number of configurations that can be populated by the molecule28) at a given temperature. Constraining the conformations of the denatured state, therefore, would lower its entropy while keeping the entropy of the native state the same. Thus, ΔSd could be lowered while raising ΔGd, which should stabilize the enzyme. We hypothesized that by intercalating enzymes in the 2D space of the galleries of layered inorganic materials, the entropy of the denatured state can be lowered, raising ΔGd. Our approach, therefore, was to use the nanoplates of the inorganic layered solid, α-Zr(IV)phosphate (α-ZrP), for enzyme intercalation and test its stability.29,30 Although minimal or no protein structural changes were observed in the cases discussed here, if there are significant structural changes on binding to the solid then the corresponding free-energy contributions need to be taken into account, and these aspects are further discussed later. This report, therefore, reviews our various efforts to control enzyme binding to α-ZrP and enhance

Enzymes bound to nanomaterials can overcome several of these limitations. Enzyme stability, for example, has been improved by constraining the enzyme between inorganic nanosheets,17−19 and the high cost of enzymes is obviated by recycling solid-bound biocatalysts.20,21 Often, enzymes bound to solids improved their enzymatic activities by more than 100%,22,23 and solid-bound enzymes sustained activities at temperatures that are close to the boiling point of water.24,25 The substrate specificity was improved upon placing them in contact with inorganic nanomaterials and expanding their activity to multiple substrates.26,27 However, there are no rational approaches to tailoring the nanobiocatalysts with desired improvements in properties, and there are no qualitative or quantitative models to predict enzyme behavior at the nanobio interfaces. In this context, we began systematic studies to evaluate the mechanism of enzyme binding to nanomaterials and factors that control bound-enzyme behavior. These data are being used to develop rational approaches to controlling the nanobio interface and bound-enzyme properties. 1.1. Enzyme Stability at the Nanobio Interface: Thermodynamic Constraints. The intrinsic stability of any enzyme is governed by its respective thermodynamic states, that is, the free-energy gap (ΔGd) between the native state (N) and the corresponding denatured state (D). This gap is generally positive for all enzymes at room temperature, and it decreases with increasing temperature as a result of increased contributions of the −TΔSd term. ΔGd reaches zero at the denaturation temperature, and enzyme denaturation is spontaneous above this temperature. If the ΔGd of the bound enzyme is increased by interactions at the nanobio interface, 14002

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Figure 1. (A) Schematic structure of α-ZrP, (B) TEM image of individual plates of α-ZrP, and (C, D) SEM images of stacks of α-ZrP.

Table 1. Key Features of Enzymes Used in This Study44,53 a

a b

enzyme/property

pI

net charge (pH 7)

hydrodynamic radius (Å)

area of cross section (Å2)b

molecular wt (kDa)

number of residues

Lys Cyt c CHT Mb Hb HRP GO BSA α-lactalbumin Tyrosinase

10.9 10.1 8.7 6.9 6.6 5.1 4.6 4.8 4.5 4.3

7 7 −1 −4 −8 0 −62 −18 −7 −8

19.8 17.2 20 20.4 32 28 40 33.7 18.8 49.1

1232 929 1256 1307 3217 2463 5026 3568 1110 7574

14.3 12.4 25 16.5 64.5 44 155 66.5 14.2 128

129 104 241 153 427 308 581 576 123 1126

Hb, hemoglobin; Mb, myoglobin; CHT, chymotrypsin; Lys, lysozyme; Cyt, cytochrome c; HRP, horseradish peroxidase; and GO, glucose oxidase. The area of the cross section of each protein is calculated from its hydrodynamic radius, considering it to be a sphere.

leaching during catalysis. Therefore, covalent binding to the solid is not required, and the nanobio interface can be readily manipulated to control the bound-enzyme affinity, structure, loading, and stability, as shown below. 2.2. Structure and Preparation of α-ZrP. A schematic structure of α-ZrP is shown in Figure 1A, which consists of 7.6Å-thick nanoplates made from a layer of Zr(IV) ions (open circles) sandwiched between two layers of phosphate anions (blue spheres). Each Zr(IV) ion is octahedrally coordinated by six oxygen atoms arising from three different phosphates. The remaining OH group of each phosphate (pink sphere) is oriented perpendicular to the metal plane, and α-ZrP nanoplates hold a very high charge density (maximum of 1 charge per 25 Å2) as a result of weakly acidic OH groups with a pKa ≈ 7. α-ZrP nanoplates (Figure 1B) stack into columns because of van der Waals interactions (Figure 1C,D). α-ZrP was prepared by the addition of aqueous ZrOCl2 solution to phosphoric acid, followed by reflux (24 h, 80 °C, eq 1).31,32 It is obtained as a dry, white powder (40−50% yield).30

its stability by constraining the conformational entropy of the corresponding denatured states. First, we describe our approaches to enzyme intercalation (Scheme 1), and then we describe specific examples as well as their degrees of success in achieving the above goals, which ultimately resulted in benign, proteinophilic interfaces for enzyme loading.

2. LAYERED INORGANIC SOLID, α-ZRP Layered α-Zr(IV)phosphate (α-Zr(HPO4)2·H2O, abbreviated as α-ZrP) was chosen for a number of reasons. α-ZrP is a stable metal phosphate with a layered structure (Figure 1A).31,32 This solid and a number of its derivatives were readily synthesized and purified in large amounts. The layered structure accommodates enzymes of any size, from very small to very large, monomeric to multimeric, and globular, cylindrical, or other shapes. Its high polarity and hydration state allow the binding of cationic or anionic and water-soluble globular enzymes, and it could preserve the native structure of the enzymes when compared to hydrophobic surfaces of other solids, which often denature the bound enzyme. The large surface area of α-ZrP (100 m2/g) and its high charge density33 of 1 negative charge per ∼25 Å2 or ∼6.6 × 10−6 mol/m2 controls binding via electrostatic and other interactions. In comparison, solids of lesser charge density, such as montmorillonite with a charge density of ∼2.0 × 10−6 mol/m2,34 are expected to make weaker charge contributions to the binding process. Gallery spacings of the enzyme/α-ZrP intercalated materials are small enough to protect the enzyme from microbial or protease degradation, but they are wide enough to permit the diffusion of reagents and substrates into and out of the galleries. The high residual charge of the α-ZrP nanoplates provides a convenient handle for monitoring enzyme binding by zeta potential studies, and the layered structure provides an elegant method for testing intercalation by powder X-ray diffraction. The negative charge field and the mechanical barrier offered by the nanoplates hold the intercalated biocatalyst without

ZrOCl 2 + PO(OH)3 → α‐Zr(HPO4 )2 ·nH 2O

(1)

2.2. Enzymes Examined and Their Characteristics. Numerous studies describing the intercalation of ions,35,36 small molecules,37−39 metal complexes,40 and organic cations41,42 in the galleries of α-ZrP have been reported. These early studies provided a strong basis for the intercalation of enzymes in the galleries of α-ZrP to test the above hypothesis of entropy control of enzyme stability by constraining enzymes between nanoplates. We have chosen a small set of representative enzymes with charges ranging from +7 to −62 (Table 1). These represent a variety of sizes, shapes, charge densities, isoelectric points (pI), different secondary/tertiary structures, catalytic functions, and stabilities.

3. ENZYME BINDING TO α-ZRP NANOPLATES The binding of positively charged enzymes to α-ZrP nanoplates is expected to be facile because of favorable electrostatic 14003

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Figure 2. (A) Exfoliation of α-ZrP stacks for enzyme binding and their reassembly, (B) powder XRDs of the enzyme/α-ZrP intercalates and pristine α-ZrP (inset), (C) strong correlation between the measured d spacings of the intercalates and the known sizes of enzymes,26 (D) Hb binding to αZrP, with the integrated heat released (closed dots) and the best fit to the data (red line).44

of the protein solution (100 μM, 20 mM NaPipes pH 7.2, 1 mM TBA) to a suspension of α-ZrP (1 mM, same buffer as protein solution) loaded into the calorimeter until the binding was saturated. The heat released or absorbed (Q) was monitored in real time and analyzed using eq 2. From the known values of the calorimeter volume (Vo), the bulk concentration of the ligand (Xt), the concentration of α-ZrP (Mt), the binding constant (Kb), the binding site size (n), the enthalpy of binding (ΔH), the entropy of binding (ΔS), and the free energy of binding (ΔG) were calculated by the single, identical binding site model. Goodness of fits and uniqueness of fits were tested analytically.

interactions between the enzyme and the anionic nanoplates. However, because of the very large sizes of enzymes (30−100 Å), direct intercalation is kinetically very slow and does not proceed at desirable rates. Hence, we considered the exfoliation of α-ZrP stacks prior to intercalation and exposing the nanoplates to the enzyme solutions for successful binding. 3.1. Intercalation and Powder X-ray Diffraction. Exfoliation of the stacks of α-ZrP by treatment with a large cation such as tetrabutyl ammonium (TBA) hydroxide was previously reported (Figure 2A),37 and we discovered the successful binding of enzymes to exfoliated nanoplates. Subsequent to binding, the enzyme-bound plates spontaneously assembled to reform stacks, resulting in enzyme/α-ZrP intercalates.43 Enzyme intercalation is expected to increase the d spacings between the nanoplates up to the diameter of the intercalated enzyme, and intercalation was established by powder X-ray diffraction data.43,26 As expected, enzyme binding resulted in strong X-ray diffraction patterns (Figure 2B), and new peaks corresponding to the intercalated enzymes have been noticed. Peaks of α-ZrP/TBA or pristine α-ZrP (Figure 2B, inset) are no longer present in these patterns, which clearly established enzyme intercalation between the nanoplates of α-ZrP. In support of this conclusion, a plot of the observed d spacings of the enzyme intercalates versus the average known size of the intercalated enzymes was linear (Figure 2C). The nonzero y intercept of 6 Å for the linear fit has been attributed to several layers of hydration at the nanobio interface, and such hydration is critical to maintaining the native structures of the intercalated enzymes. 3.2. Binding Studies by Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) was used to investigate enzyme binding to the nanoplates, and ITC provides a direct measure of the binding enthalpies and binding stoichiometries in a model-independent manner. When used with specific binding models, one could estimate binding enthalpies, entropies, affinity constants, and binding site sizes as well. The energetics of enzyme binding to the nanoplates were measured with a nanocalorimeter by adding aliquots (2−4 μL)

Q=



⎡ nM tΔHVo ⎢ Xt 1 ⎢1 + nM + nK M 2 t b t ⎣ ⎤ 2 ⎛ 4X t ⎥ X 1 ⎞ ⎟ − ⎜1 + t + nM t nKbM t ⎠ nM t ⎥ ⎝ ⎦

(2)

The titration of a solution of Hb into a suspension of exfoliated α-ZrP nanoplates (Figure 2A), for example, was accompanied by heat release (Figure 2D, exothermic). The ITC data were fitted by single, noncooperative, identical site model, and the best fit to the observed data (red line) indicated a binding constant, Kb, of 2.4 ± 0.3 × 106 M−1 and a binding site size of ∼450 phosphates per Hb.44 The Kb and the binding site size obtained from ITC matched those obtained from centrifugation binding studies. This confirmed the validity of the binding model and provided useful insight into the thermodynamics of Hb binding. For example, exothermic binding is accompanied by significant entropy loss (ΔS = −50 kcal/Kmol), and the origin of this loss is explained later in this article. Next, we examined the structure and activities of bound enzymes. 3.3. Binding Capacity (Loading) on α-ZrP. The loading of enzymes on nanoplates is limited by a number of attributes of the nanobio interface, and loading is important in biocatalytic applications because higher loadings reduce the bioreactor size and setup costs. Loadings of various enzymes on 14004

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α-ZrP have been measured by centrifugation binding studies where the bound enzyme is separated from the free enzyme. Maximal loadings obtained for enzyme/α-ZrP (g/g) with Lys, Cyt c, CHT, Mb, Hb, and GO were 1.4, 1.3, 1.9, 0.48, 2.25, and 0.44, respectively. These loadings are much higher than normally noticed with other solid supports. Although positively charged enzymes (Lys, Cyt c) had higher loadings on the negatively charged α-ZrP than did negatively charged enzymes (Mb, GO), Hb had an unusually higher loading of 2.25 (w/w). These loadings are used to calculate the average areas occupied by the enzymes in the galleries of the intercalated nanobiocatalyst. Cyt c binding studies, for example, indicated that 1 enzyme molecule occupied 435 phosphate groups on the α-ZrP nanoplate. This loading translates to a footprint of ∼100 × 100 Å2 per Cyt c, a value that is much larger than the known area of the cross-section of Cyt c (929 Å2).43 These data suggest that there are large distances between adjacent enzyme molecules, and such voids would facilitate the diffusion of reagents into or out of the galleries. Such access to the bound enzyme is also essential to the retention of intercalated enzyme activities. However, the denaturation of the bound enzyme would occupy a larger area on the nanoplates, but such denaturation would be easy to detect in spectral studies where the enzyme structure can be readily monitored, as described below. 3.4. Bound-Enzyme Structure. Enzyme−nanomaterial interactions often result in the distortion of the bound enzyme structure,45 which are not desirable because these could result in the deactivation of the enzyme. Structural changes of enzyme/α-ZrP samples were monitored by infrared absorption (FTIR) and circular dichroism (CD) studies. For example, amide vibrational bands (I, II, and III) of enzymes are highly sensitive to enzyme structural changes,46 and FTIR spectra of enzyme/α-ZrP intercalates indicated extensive structure retention (Table 2). The amide I/II bands of Mb/α-ZrP are nearly the same as those of Mb, and similar observations were noted for the other samples.

medium-intensity negative bands at 210 and 222 nm, whereas a broad negative peak at 212 nm is a characteristic of β sheets.47 Random coils result in a strong, sharp, negative peak at 195 nm. Hence, the CD spectra are often useful in assessing bound enzyme structure.48 The CD band positions of the above samples were examined, and they indicated the extensive retention of native-like structures (Table 2). Such a high degree of structure retention is unusual but bodes well for the retention of enzymatic activities of these biocatalytic nanomaterials. 3.5. Improved Catalytic Activities. The retention of enzymatic activities of the enzyme at the nanobio interface has several stringent requirements such as (1) substantial retention of the active-site structure; (2) good accessibility to the active site; (3) facile diffusion of the substrate to the active site and release of the product from the active site into the solution; and (4) sufficient flexibility of enzyme structure such that the active site can undergo conformational changes that are necessary for catalytic activity. Conversely, activity studies probe subtle features of the nanobio interface of the bound enzyme and its active-site structure on the molecular level. Specific rates, the Michaelis constant (Km), and the maximum velocity of the reaction (Vmax) were determined (Table 3), and all biocatalysts retained significant activity with some of them showing higher activities than do the free enzymes. In the cases of GO/α-ZrP and CT/α-ZrP, for example, the activities increased to 125 and 130% of the activities of the corresponding free enzymes, respectively. However, the activities of lysozyme, CHT, and Mb intercalates are very close to those of the corresponding free enzymes. These observations are remarkable because enzyme binding to solid substrates often reduces their enzymatic activity.49,50 An observed increase in the Vmax in the case of Hb/α-ZrP and unchanged Km are favorable outcomes as well. When boundenzyme structures are close to those of the unbound enzymes, biocatalysts retain their native-like activities, but still higher activities are highly desirable and possible. The above activity data clearly demonstrate that the diffusion of the substrate to the active sites of the intercalated enzymes is facile. The retention of native-like activities is essentially due to the extensive retention of their native-like structures. Although the above criteria ensure high activities, limiting the value of Km is tricky and difficult, although an example is shown later. The structure retention, flexibility of the bound protein conformations, access to the active site, and release of the product into the solution are all important steps in maintaining the boundenzyme activity. 3.6. Enhanced Substrate Specificity. We also assessed the selectivities of the intercalated biocatalysts in one particular case. Most enzymes are specific to one or a few substrates, and broadening the selectivity will be crucial to wider use in the laboratory or in industry. However, some enzymes are

Table 2. Amide I/II Peak Positions and Helical Contents from FTIR and CD Spectra26 enzyme

amide I/II free

amide I/II bound

CD bands (nm), free

CD bands (nm), bound

Hb Mb Lys GO

1654/1521 1651/1530 1650/1521 1645/1538

1653/1521 1651/1530 1650/1521 1645/1538

210/222 210/222 207/223 210/218

210/222 210/222 207/223 210/218

Enzymes display myriad well-defined 3D structures built from a few secondary structural motifs. For example, α-helixes, β-sheets, and random coils dominate the structural elements of enzymes. These structural details have particular CD bands. For example, α-helices show strong positive peaks at 190 nm and

Table 3. Specific Activities, Km, and Vmax of Enzyme/α-ZrP Intercalates26 specific activity (μM−1 s−1) enzyme Mb Lys Hb CHT GO

free 3.3 11.0 1.3 3.1 2.0

× × × × ×

10−3 10−3 10−2 10−1 102

bound 2.0 9.4 3.8 4.0 2.5

× × × × ×

Vmax (μM s−1)

Km (mM) 10−3 10−3 10−2 10−1 102

free

bound

free

bound

1.4 0.5 0.103

1.6 0.5 0.112

0.08 0.63 0.0034

0.04 0.63 0.0042

0.8

2.5

37

42

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constant, ΔG depends solely on ΔS, and according to SNSDDS, when ΔΔS is lowered, a higher temperature is required to denature the enzyme. Hence, the stability of the enzyme could be improved. DSC provides a direct measure of the enthalpy of denaturation (ΔHd), the denaturation temperature (Tm), and the heat capacity change accompanying the denaturation process (ΔCp) in a model-independent manner. When denaturation is reversible, ΔGd and ΔSd could also be determined using particular denaturation models. The DSC thermograms of intercalated enzymes showed extensive broadening, and denaturation continued well beyond the corresponding Tm of the free enzyme. Thus, a fraction of intercalated enzyme was found to be more stable than the corresponding free enzyme in almost every case.51 Extensive broadening of the thermograms could be due to conformational heterogeneity, multipoint contact of the enzyme with the solid, heterogeneity in the population of the binding sites, multistep kinetics of denaturation, or/and enzyme aggregation. Thus, DSC data analysis is complicated, but a significant fraction of the intercalated enzyme had a higher ΔHd (Figure 3A). Because

nonselective, and improving their selectivity will be vital for selective transformations. Mb, for example, is not an enzyme, but it efficiently catalyzes the oxidation of electron-rich phenols by hydrogen peroxide and has little or no selectivity. The substrate specificities and rates of oxidation by Mb/α-ZrP and Mb were determined with a number of ortho, para, or meta isomers of substituted phenols, and the ratios of these rates are given in Table 4. Values >1 represent accelerated rates for the intercalated biocatalyst compared to those for the unbound Mb, whereas values Ca(II) > Cr(III) > Mg(II) ≫ H(I) > Na(I). Therefore, metal ion charge is not the only factor controlling its ability to promote GO binding to α-ZrP. The coordination of metal ions to the surface carboxyl groups of the enzyme and the phosphate surface groups of the solid also played a major role. For example, Zr(IV) is by far the most oxophilic and phospholic among these,56 and it promoted the binding the most, followed by Ca(II) and then Cr(III). Ca(II) was much more effective than Mg(II) because Ca(II) is more oxophilic than Mg(II). In contrast to the strong improvements in GO binding by metal ions, the binding of Hb, which has fewer COOH groups on its surface than GO,55 improved only marginally. It increased from ∼80 to ∼97% by the addition of 0.5 mM Zr(IV), and surprisingly, higher Zr(IV) concentrations inhibited Hb binding. This latter observation was attributed to the requirement that the metal should bind to both the protein and the solid with high affinity, but if it binds to α-ZrP better than to the enzyme, then enzyme binding may be

5. BUILDING ON THE ICPB MODEL The ICPB model was exploited to control enzyme binding to α-ZrP. According to the ICPB model, for example, the binding of negatively charged enzymes to α-ZrP is accompanied by the sequestration of cations at the enzyme/solid interface so that excess negative charge at the interface is balanced. This proposition predicts that metal ions that have a high affinity for the solid should promote the binding of the anionic enzymes.55 Thus, particular metal ions are predicted to serve as excellent metal glues (Figure 5A) for the binding of anionic enzymes to α-ZrP. 5.1. Modulation of Enzyme Binding with Metal Ion Glues. A number of metal ions were tested for their ability to function as metal glues to bind enzymes to α-ZrP, as predicted 14009

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Figure 7. (A) Enzyme cationization to enhance binding. (B) Agarose gels of protein−TEPA conjugates. Lane 1 contains GO, and lanes 2 and 3 contain GO-TEPA (4 h reaction) and GO-TEPA (12 h reaction), respectively. Lane 4 contains Hb, and lanes 5 and 6 contain Hb-TEPA (8 h reaction) and Hb-TEPA (12 h reaction), respectively. (C) Binding isotherms of GO and GO-TEPA with α-ZrP (10 mM phosphate, 50 mM NaCl, pH 7.2). (D) Scatchard plots for GO and GO-TEPA binding to α-ZrP. (E) Comparison of enzyme activities.53

and exfoliated α-ZrP (6 mM) promoted DNA binding as a function of increasing Hb concentration (Figure 6B), and the binding of DNA indicated a bell-shaped curve as a function of Hb concentration. DNA did not bind to the solid in the absence of Hb, and >1 nmol of DNA was bound per every 100 nmol of α-ZrP. This approach of using Hb as a glue provided a facile method of intercalating various nucleic acids in the inorganic layered materials, which is not otherwise possible. The Hb/DNA/α-ZrP ternary complex was characterized by SEM, TEM, XRD, CD, and activity studies. The SEM and TEM micrographs indicated a 48 ± 1 Å spacing between nanoplates, and this value was verified by powder XRD measurements that indicated a d spacing of ∼40 Å (data not shown). Unexpectedly, the intercalation of DNA improved the Hb structure retention and its catalytic activities when compared to those of Hb/α-ZrP. The CD spectrum of Hb/ DNA/α-ZrP, for example, was nearly indistinguishable from that of Hb.58 The peroxidase-like activity of Hb/DNA/α-ZrP steadily increased as a function of DNA concentration to a maximum of 1.6 × 107 s−1 per mole of Hb (Figure 6C), which is much greater than that of Hb/α-ZrP under the same conditions of pH, temperature, and substrate concentration.58 Similar observations were noted when Hb was replaced with Mb. Thus, Hb and Mb served as protein glues to improve the binding of DNA to α-ZrP, and DNA served as an unusual cointercalant to enhance the behavior of Hb and Mb. Biological

inhibited. Interestingly, Zr(IV) enhanced the maximal loading of Hb from 225 to 400% (w/w), making it one of the highest loadings noted for a biocatalyst. Hb binding followed the order Zr(IV) > H(I) > Mg(II) > Na(I) > Ca(II) > Cr(III), showing the importance of the chemical nature of the ion rather than its charge as the governing factor for the metal glues. H(I) was more effective in promoting Hb binding α-ZrP than trivalent Cr(III) or divalent Ca(II) (Figure 5B), which is consistent with the buffer ion dependence studies described before. Therefore, the effect of the metal ion depends on its ability to bind to the nanobio interface rather than just to the solid or to the enzyme surface. This conclusion is consistent with the ICPB model, and the ensuing thermodynamic effect is more than electrostatic. However, careful selection of the metal ion is required because of the inherent risks of certain metal ions that could inhibit enzyme activities,57 but α-ZrP might compete for these ions and weaken such inhibition. 5.2. Proteins as Soft Counter Ions. Along the above lines of deduction, the ICPB mechanism also predicts that proteins bind strongly to the solid and the biomolecule of interest could serve as a protein glue. This aspect was tested,58 where Hb served as a glue to bind anionic DNA and RNA to α-ZrP (Figure 6A). Anionic DNA and RNA have only weak affinities for anionic α-ZrP, but the presence of suitable proteins that bind to the DNA/inorganic interface should promote binding (Figure 6A). The addition of Hb to a mixture of calf thymus DNA (80 μM) 14010

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Figure 8. (A) Biophilization of α-ZrP by with highly cationized BSA (cBSA, BSA-TETA(+27)), followed by enzyme binding. (B) Binding isotherm of cBSA with α-ZrP (6 mM). (C) CD spectra of BSA (blue), cBSA (red), and bZrP (cBSA/ZrP). (D) Enhanced binding of GO to bZrP (blue) when compared to binding to ZrP (black) (unpublished data).

confirmed by mass spectrometry, and these samples provided an excellent means to test the ICPB mechanism. Binding studies with GO-TEPA(+20) (50 μM) indicated that the amount of GO-TEPA bound is much greater than that of GO (Figure 7C) under the same conditions of pH, temperature, ionic strength, and α-ZrP concentration.53 An analysis of the binding data indicated kb = (1.1 ± 0.2) × 107 M−1, which is 400 times larger than that of GO, demonstrating the strong role of charged residues at the nanobio interface. Hb-TEPA(+16), however, showed a 26-fold increase in affinity for α-ZrP (kb = 1.4 × 107 M−1) when compared to that of Hb (kb = 4.4 × 105 M−1) under the same conditions of pH, ionic strength, and temperature. These observations support the hypothesis that inserting additional positive charges or polar groups at the nanobio interface can enhance the affinity by orders of magnitude, consistent with the predictions of the ICPB mechanism.53 Enhanced binding was also achieved without a significant loss of enzyme structure or activity. The CD spectrum of GOTEPA/α-ZrP showed only a 15−20% loss in structure when compared to that of pristine GO (Figure 7D) but was much greater than that of corresponding GO/α-ZrP.59 Activities of GO-TEPA/α-ZrP samples showed an ∼2.5-fold improved activity when compared to that of GO/α-ZrP, whereas HbTEPA/α-ZrP retained 76% it its Hb activity (Figure 7E). Hence, the ICPB mechanism provided useful insights into controlling and improve affinities in a systematic manner without compromising activity.

counterions thus appear to be excellent at improving enzyme affinities and activities at the nanobio interfaces.

6. CONTROL OF ENZYME BINDING VIA CHARGE TUNING: CHEMICAL MODIFICATION The ICPB model predicts that enzyme binding to α-ZrP depends on the number of charged residues on the enzyme surface and not necessarily on the net charge on the enzyme. Therefore, we systematically engineered positively charged side chains on enzyme surfaces via chemical modification (Figure 7A) and tested their binding behavior.59 In simple terms, positively charged groups placed at the nanobio interface could enhance enzyme binding. This hypothesis was tested by introducing polyamine chains onto the enzyme surface via chemical modification and testing the binding affinities of the modified enzymes. The amidation of the enzyme-surface COOH groups with polyamines converts them into the corresponding amide polyamines, thereby neutralizing the negative charge of the ionized COOH group and also providing additional protonatable amino groups. Depending on the pH of the medium, the charge could potentially change from −1 to +n for each amidation event, with a maximum change in charge by n + 1 electrostatic units. Furthermore, the enzyme charge can be adjusted over a wide range by controlling the extent of amidation, as desired. The COOH groups of negatively charged GO (pI 4.6) and Hb (pI 6.8), for example, were chemically modified with tetraethylenepentamine (TEPA) by EDC chemistry to obtain enzyme charge ladders (Figure 7B).53 The progress of the chemical modification was followed by agarose gel electrophoresis in which the unmodified anionic enzymes moved toward the positive electrode whereas the cationized enzymes moved toward the negative electrode (Figure 7B). The cationized samples were named on the basis of the enzyme, polyamine, and net charge. GO-TEPA(+20), for example, indicated GO modified with TEPA with a net charge of +20. The chemical modification and charge reversal were also

7. BIOPHILIZATION OF α-ZRP AND EXPANDING ITS UTILITY The above concepts of enhanced affinities of cationized proteins for α-ZrP and the use of protein glues to bind nucleic acids were combined to develop a general method to control enzyme binding to α-ZrP systematically. This alternative could be useful in other respects as well. The chemical modification of each enzyme to adjust its charge, for example, could be cumbersome or reduce its biological activity, requiring additional purification steps. However, developing a general 14011

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Figure 9. (A) Schematic representation of binding of Hb-PAA conjugates bZrP nanosheets. (B) Binding of Hb(2)-PAA(1)-6 to bZrP (purple line) and Hb(2)-PAA(1)-6 (beige line) binding to α-ZrP, (C) Comparison of activities of Hb and GO intercalated using particular strategies discussed in this review (unpublished data).

temperature (40−80 °C), GO/bZrP showed a sharp, clean, single-step denaturation that is noted for the first time for any protein bound to α-ZrP. The increase in Tm and the sharp thermal transition provided support for the claim that the αZrP surface is now more biocompatible by the adsorption of BSA-TETA(+27), or this behavior could be referred to as proteinophilic. 7.1. Exploitation of bZrP for Protein Binding. Cationic bZrP should readily bind a number of anionic biomolecules or biomolecular−protein conjugates (Figure 9A), and the cBSA layer should provide a biophilic surface for favorable interactions with the bound enzyme whereas the layered structure of the inorganic could enhance the thermal stability of the intercalated enzyme−-polymer conjugate. We previously prepared a number of Hb-poly(acrylic acid) covalent conjugates (Hb-PAA) by EDC chemistry,63,64 and these provided new opportunities to test the utility of bZrP. Our hypothesis is that the anionic Hb-PAA conjugates would bind favorably to bZrP with extensive structure retention, as in the case of GO/bZrP, whereas the layered structures of the intercalates would enhance the thermal stabilities of the conjugates. High stability/activity could be important for fuel cell or high-temperature biocatalysis applications of intercalated enzymes, and bZrP provided unique opportunities to enhance the affinity, activity, and stability. The Hb-PAA conjugates consisting of Hb to PAA mole ratios of 2:1 were prepared at pH 6 by EDC chemistry, and the resulting conjugates are denoted as Hb(2)-PAA(1)-6. These polymer conjugates bound avidly to bZrP (Figure 9B, purple curve). At 60 μM Hb(2)-PAA(1)-6, nearly all of the conjugate was bound to bZrP whereas only half as much has been bound to α-ZrP (beige curve). A maximum loading of Hb(2)-PAA(1)6 for bZrP was 240% (w/w), but it was limited to 125% (w/w) with α-ZrP. Thus, the underlying cBSA layer enhanced the binding due to favorable interactions at the interface. bZrP also improved the Hb(2)-PAA(1)-6 activities (Figure 9C, purple bar), which is nearly equal to that of Hb. Further studies are in progress to test bZrP for enzyme binding, stability enhancements, and activity studies. Thus, bZrP expanded the utility of α-ZrP for binding anionic proteins and anionic protein−polymer conjugates, and it provided new opportunities to implement this strategy of controlling affinities as well as enzyme behavior by the rational manipulation of the nanobio interface.

method to enhance the binding of a given enzyme without its chemical modification would be highly attractive and efficient. If successful, this alternative approach could be adapted to other solid surfaces as well, and hence these experiments were considered to be important in generating novel, facile, economic, benign, and convenient approaches to the binding of enzymes and proteins to almost any solid surface. Our strategy has been to cationize BSA (pI = 4.7)60 by reacting its COOH groups with triethylenetetraamine (TETA) using EDC chemistry and to bind supercharged BSA-TETA to α-ZrP and systematically control the net charge of α-ZrP platelets for enzyme binding (Figure 8A). Highly cationized BSA-TETA(+27) indicated strong binding to α-ZrP (Figure 8B) with a high binding constant, kb = 1.5 × 107 M−1, whereas BSA indicated a very poor affinity for α-ZrP. BSA-TETA(+27) showed the complete retention of its structure (Figure 8B, red dashed line), but binding to α-ZrP resulted in the complete loss of its secondary structure (Figure 8B, black line). The BSATETA(+27)/α-ZrP nanoplates are positively charged, as indicated by the zeta potential studies, and they are suitable for binding anionic enzymes with high affinity. The BSATETA(+27) layer is expected to provide a benign surface and enhance bound-enzyme structure, activity, and stability. This approach differs from the layer-by-layer technique61 where the binding of a single layer of modified protein is used. The variety of amino acid side chains present on cBSA provides a microcosm of interactions for the benign binding of the enzyme, mimicking a cellular-like environment that is distinct from that of an artificial polymer or polyelectrolyte. The BSA-TETA(+27)/α-ZrP nanoplates, termed here as biophilic-ZrP (bZrP), were tested for the binding of negatively charged enzymes such as GO. The binding of GO to bZrP resulted in high loadings of ∼700% (w/w) of GO to bZrP (highest known so far), whereas GO bound very poorly to pristine α-ZrP (Figure 8D). Ideally, a biophilic surface should bind and stabilize the bound biomolecules, and we tested this aspect by monitoring the thermal denaturation of GO/bZrP by differential scanning calorimetry (DSC) (data not shown). The denaturation temperature of GO/bZrP was found to be 72 °C, which is substantially higher than that of GO (60 °C) under the same conditions of ionic strength and pH.62 In addition to the increases in Tm, the DSC thermograms also showed much improved characteristics. Unlike in previous studies, where the direct intercalation of enzymes in α-ZrP resulted in broad thermograms over a wide range of 14012

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8. CONCLUDING REMARKS The major issue in this field is how to predict the properties of an enzyme residing at a given nanobio interface. The enzyme properties of interest are affinity, structure retention, activity, and thermal stability. The above studies show that binding enthalpies and entropies of many α-ZrP enzymes can be predicted from the enzyme charge. The binding enthalpies and entropies can be combined to predict the binding affinities (ΔG) with a reasonable measure of confidence. Given the issues of enthalpy−entropy compensation for enzyme binding and the contributions of enzyme structure loss to the observed enthalpies, the overall affinities of enzymes may not be accurately predictable from their net charge. Therefore, we conclude that systematic thermodynamics studies can be powerful predictive tools for affinities. There may be other indicators that can predict enzyme affinity, but presently there are no known methods of such prediction, experimental or theoretical. Another major conclusion is that the fundamental basis for this thermodynamic control is the basic requirement of charge neutralization when the surfaces are electrically charged. That is, when the two surfaces meet during the binding process, the charge density of the guest surface needs to match the charge density of the host surface, and this charge matching requires either the release of ions or the recruitment of ions of appropriate charge, number, and affinity. Thus, binding affinities could be systematically controlled by the nature and extent of ions present in the solution. When the charge density on the enzyme matches that of the nanosheets, the binding is still governed by the ICPB mechanism because each charged surface carries counterions. However, when there is a charge mismatch, greater numbers of ions will be involved in the mechanism. This is because the mismatch requires the release or sequestration of additional ions. The ICPB mechanism provided excellent control over enzyme affinities via the control of the concentrations and compositions of other participating species that are present. Using this insight, we were able to drive the intercalation of nucleic acids and other anionic proteins into anionic α-ZrP, which is otherwise not possible. These led to the discovery of metal glues and later protein glues and polymer glues for enzyme binding to α-ZrP. We suspect that metal glues, where the metal ion drives the binding, could provide a strong, predictable approach to controlling the affinity. This hypothesis needs to be tested in future studies. Along these lines, the discovery of protein glues and polymer glues is of major importance and provides powerful strategies for controlling enzyme binding as well as the retention of bound enzyme structure at the nanobio interface. For example, Hb has high affinities for both DNA and α-ZrP, and this fact provided an opportunity to use Hb as a glue to bind nucleic acids to α-ZrP. This tactic provided an excellent avenue to enhance the binding of anionic guests to anionic α-ZrP with the retention of enzyme structure and activity. Inspired by the above approach, it is hypothesized that when a sacrificial protein is properly modified, it could serve as a protein glue to bind an enzyme of interest to a particular solid with high affinity and structure retention. The use of cBSA as a protein glue has three distinct advantages: (1) each enzyme need not be chemically modified individually to promote binding to α-ZrP, and cBSA can be used to promote the

binding of almost any anionic enzyme; (2) the adsorption of cBSA on α-ZrP provides a more biocompatible underlayer to which the enzyme could be bound; and (3) if this approach is successful with α-ZrP, then cBSA may be used to biophilize a number of other inorganic nanomaterials such as metal oxides, metal nanoparticles, or silica nanoparticles. Several examples are presented above that substantiate the proposed hypothesis of the protein glue. One example of a polymer glue was also demonstrated as a proof of principle where the protein binding was controlled by the polymer content. This approach is also potentially universal and could provide stringent control, especially when used with metal glues. Although these studies are focused on α-ZrP, the physical insights are applicable to other nanobio interfaces, but this needs to be tested in future studies. Using these methods, one could envision the biophilization of nanomaterials for the binding of anionic or cationic enzymes with a high degree of predictability/control. The above studies show the successful intercalation of 10 different enzymes and proteins in the galleries of α-ZrP. In addition, we also examined carboxymethyl and carboxypropyl analogues of α-ZrP for protein binding and biocatalysis, which are not discussed here. A majority of these bound enzymes have indicated the significant retention of structures and activities, and some indicated improved selectivities26 and recyclability.62 In general, these inorganic solids appear to be benign for enzyme loading and provided biocatalysts with improved properties. One potential approach to enhancing biocatalytic activities further would be to intercalate two or more enzymes that work in tandem, speeding up the overall rates. This is yet to be tested systematically. Inorganic materials continue to have a strong impact on enzyme loading, bioreactors, and biomaterials that come in contact with proteins or enzymes. The thermodynamic and kinetic arguments provided here should also be applicable to all other solids such as metal phosphates, oxides, semiconductors, and graphitic and related materials. The primary reason for this expectation is that the above principles of enzyme binding and manipulation of the enzyme/inorganic interface does not rely on the molecular structure of α-ZrP but rather relies on its charge, layered topology, and hydrophilic nature. One critical advance that is required would be to quantify the influence of lower dimensions of space on protein stability. The above investigations support this stability hypothesis, but it needs to be tested further. Another fundamental issue to address is what functional groups at the biomolecule/solid interface are the best for the retention of structure and activity. Only limited data are currently available on this topic, but the use of systematically modified protein and polymer glues can begin to unravel these details. One major challenge would be to provide biocatalysts that can function efficiently at high temperatures and in organic media for applications in organic synthesis, biofuel cells, and the industrial production of fine chemicals. Although two examples of high-temperature biocatalysis are presented here, there is great potential for future work. These fundamental and practical aspects of the enzyme/solid interfaces are likely to be intensely investigated in future studies. The general approaches described above to control the proteinophilicity also apply to biosensing and drug-delivery applications where enzyme/solid and protein/solid interfaces are important. We hope that further evaluation of these interactions on the molecular level will continue to provide a 14013

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good foundation for designing new and improved functional inorganic biomaterials for future applications.



REFERENCES

(1) Jia, H.; Zhu, G.; Wang, P. Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility. Biotechnol. Bioeng. 2003, 84, 406−414. (2) Hartmann, M.; Jung, D. Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. J. Mater. Chem. 2010, 20, 844−857. (3) Cassimjee, K. E.; Kourist, R.; Lindberg, D.; Wittrup Larsen, M.; Thanh, N. H.; Widersten, M.; Bornscheuer, U. T.; Berglund, P. Onestep enzyme extraction and immobilization for biocatalysis applications. Biotechnol. J. 2011, 6, 463−469. (4) Darder, M.; Aranda, P.; Ruiz-Hitzky, E. Bionanocomposites: a new concept of ecological, bioinspired, and functional hybrid materials. Adv. Mater. 2007, 19, 1309−1319. (5) Taylor-Pashow, K. M. L.; Della Rocca, J.; Huxford, R. C.; Lin, W. Hybrid nanomaterials for biomedical applications. Chem. Commun. 2010, 46, 5832−5849. (6) Santiago-Berríos, M. E. B.; Declet-Flores, C.; David, A.; Borrero, S.; Vélez, M. M.; Díaz-Díaz, A.; Guadalupe, A. R.; Colón, J. L. Direct intercalation of bis-2,2′,2″,6-terpyridylcobalt(iii) into zirconium phosphate layers for biosensing applications. Langmuir 2012, 28, 4447−4452. (7) Thomson, A. J.; Gray, H. B. Bio-inorganic chemistry. Curr. Opin. Chem. Biol. 1998, 2, 155−158. (8) Daniel, R. M.; Danson, M. J. A new understanding of how temperature affects the catalytic activity of enzymes. Trends Biochem. Sci. 2010, 35, 584−591. (9) Lindberg, D.; de la Fuente Revenga, M.; Widersten, M. Temperature and pH dependence of enzyme-catalyzed hydrolysis of trans-methylstyrene oxide. A unifying kinetic model for observed hysteresis, cooperativity and regioselectivity. Biochemistry 2010, 49, 2297−2304. (10) Silver, M. S.; James, S. L. T. Enzyme-catalyzed condensation reactions which initiate the rapid peptic cleavage of substrates. 1. How the structure of an activating peptide determines its efficiency. Biochemistry 1981, 20, 3177−3182. (11) Mahler, H. R.; Douglas, J. Mechanisms of enzyme-catalyzed oxidation-reduction reactions. I. An investigation of the yeast alcohol dehydrogenase reaction by means of the isotope rate effect. J. Am. Chem. Soc. 1957, 79, 1159−1166. (12) Richard, J. P. Acid-base catalysis of the elimination and isomerization reactions of triose phosphates. J. Am. Chem. Soc. 1984, 106, 4926−4936. (13) Kawasaki, M.; Asano, Y.; Katayama, K.; Inoue, A.; Hiraoka, C.; Kakuda, H.; Tanaka, A.; Goto, M.; Toyooka, N.; Kometani, T. Asymmetric synthesis of 2-substituted 4-chromanones using enzymecatalyzed reactions. J. Mol. Catal. B: Enzym. 2008, 54, 93−102. (14) Hastings, C. J.; Pluth, M. D.; Bergman, R. G.; Raymond, K. N. Enzyme like catalysis of the Nazarov cyclization by supramolecular encapsulation. J. Am. Chem. Soc. 2010, 132, 6938−6940. (15) Liese, A.; Hilterhaus, L. Evaluation of immobilized enzymes for industrial applications. Chem. Soc. Rev. 2013, 42, 6236−6249. (16) Bommarius, A. S.; Paye, M. F. Stabilizing biocatalysts. Chem. Soc. Rev. 2013, 42, 6534−6565. (17) Mudhivarthi, V. K.; Bhambhani, A.; Kumar, C. V. Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts. Dalton Trans. 2007, 47, 5483−5497. (18) Gao, L.; Gao, Q.; Wang, Q.; Peng, S.; Shi, J. Immobilization of hemoglobin at the galleries of layered niobate HCa2Nb3O10. Biomaterials 2005, 26, 5267−75. (19) Darder, M.; Aranda, P.; Ruiz-Hitzky, E. Bionanocomposites: a new concept of ecological, bioinspired and functional hybrid materials. Adv. Mater. 2007, 19, 1309−1319. (20) Ansari, S. A.; Husain, Q. Potential applications of enzymes immobilized on/in nano materials. Biotechnol. Adv. 2012, 30, 512−523. (21) Oh, J.-M.; Biswick, T. T.; Choy, J.-H. Layered nanomaterials for green materials. J. Mater. Chem. 2009, 19, 2553−2563.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Inoka K. Deshapriya received her B.Sc. degree in chemistry from the University of Peradeniya, Sri Lanka, in 2006. She is currently pursuing her Ph.D. with Prof. Challa Vijaya Kumar at the Department of Chemistry of the University of Connecticut. Her current research is focused on biocatalytic nanomaterials and nanoparticles for biocatalysis and cellular uptake.

Challa Vijaya Kumar received his M.Sc. from Osmania University Hyderabad and his Ph.D. from the Indian Institute of Technology Kanpur in 1982. He was a postdoctoral fellow at The University of Notre Dame for 2 years and worked as a research associate at Columbia University for 4 years. He is currently a professor of biological and physical chemistry at the University of Connecticut. His research interests include chiral protein photoscissors, DNA/RNA binders, enzyme−polymer conjugates, biocatalytic nanomaterials, DNA-based artificial antenna systems, photochemical water splitting, and cancer biomarker detection.



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ACKNOWLEDGMENTS

We thank the National Science Foundation (DMR-1005609) and the University of Connecticut for generous financial support. 14014

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(22) Yiu, H. H. P.; Wright, P. A. Enzymes supported on ordered mesoporous solids: a special case of an inorganic-organic hybrid. J. Mater. Chem. 2005, 15, 3690−3700. (23) Han, Y.-J.; Watson, J. T.; Stucky, G. D.; Butler, A. Catalytic activity of mesoporous silicate-immobilized chloroperoxidase. J. Mol. Catal. B: Enzym. 2002, 17, 1−8. (24) Kumar, C. V.; Chaudhari, A. Unusual thermal stabilities of some enzymes and enzymes bound in the galleries of layered αZr(IV)phosphate/phosphonates. Microporous Mesoporpus Mater. 2003, 57, 181−190. (25) Kallury, K. M. R.; Lee, W. E.; Thompson, M. Enhanced stability of urease immobilized onto phospholipid covalently bound to silica, tungsten and fluoropolymer surfaces. Anal. Chem. 1993, 65, 2459− 2467. (26) Kumar, C. V.; Chaudhari, A. Enzymes immobilized at the galleries of layered α-zirconium phosphate: structure and activity studies. J. Am. Chem. Soc. 2000, 122, 830−837. (27) Singh, R.; Tiwari, M.; Singh, R.; Lee, J.-K. From enzyme engineering to immobilization: promising strategies for the upgrade of industrial enzymes. Int. J. Mol. Sci. 2013, 14, 1232−1277. (28) Wang, J.; Purisima, E. O. Analysis of thermodynamic determinants in helix propensities of nonpolar amino acids through a novel free energy calculation. J. Am. Chem. Soc. 1996, 118, 995− 1001. (29) Kumar, C. V.; Chaudhari, A. High temperature peroxidase activities of HRP and hemoglobin in the galleries of layered Zr(IV)phosphate. Chem. Commun. 2002, 20, 2382−2383. (30) Jagannadham, V.; Bhambhani, A.; Kumar, C. V. Enzyme annealing: thermal treatment of met-hemoglobin bound to αzirconium phosphate/phosphonates results in initial denaturation followed by recovery of activity and structure. Microporous Mesoporous Mater. 2006, 88, 275−282. (31) Clearfield, A. Group IV phosphates as catalysts and catalyst supports. J. Mol. Catal. 1984, 27, 251−262. (32) Alberti, G. Syntheses, crystalline structure and ion-exchange properties of insoluble acid salts of tetravalent metals and their salt forms. Acc. Chem. Res. 1978, 11, 163−170. (33) Kim, H.-N.; Keller, S. W.; Mallouk, T. E.; Schmitt, J.; Decher, G. Characterization of zirconium phosphate/polycation thin films grown by sequential adsorption reactions. Chem. Mater. 1997, 9, 1414−1421. (34) Sperry, J. M.; Peirce, J. J. Ion exchange and surface charge on montmorillonite clay. Water Environ. Res. 1999, 71, 316−322. (35) Rapôso, C. M. D. O.; Eon, J. G. On the nature of sandwiched chromium complexes in exchanged α -zirconium phosphate. J. Mater. Res. 2002, 5, 421−426. (36) Tindwa, R. M.; Ellis, D. K.; Peng, G.-Z.; Clearfield, A. Intercalation of n-alkylamines by alpha-zirconium phosphate. J. Chem. Soc., Faraday Trans. 1 1985, 81, 545−552. (37) Garcia, M. E.; Naffin, J. L.; Deng, N.; Mallouk, T. E. Preparativescale separation of enantiomers using intercalated α-zirconium phosphate. Chem. Mater. 1995, 7, 1968−1973. (38) Kumar, C. V.; Asuncion, E. H.; Rosenthal, G. Formation of static anthracene excimers at hydrophobic phosphate surfaces. Microporous Mater. 1993, 1, 123−129. (39) Kumar, C. V.; Chaudhari, A. Probing the donor and acceptor dye assemblies at the galleries of α-zirconium phosphate. Microporous Mesoporous Mater. 2000, 41, 307−318. (40) Rosenthal, G. L.; Caruso, J. Photochemical behavior of metal complexes intercalated in zirconium phosphate. J. Solid State Chem. 1991, 93, 128−133. (41) Kumar, C. V.; Williams, Z. J. Supramolecular assemblies of tris(2,2′-bipyridine)ruthenium(ii) bound to hydrophobically modified α-zirconium phosphate: photophysical studies. J. Phys. Chem. 1995, 99, 17632−17639. (42) Martí, A. A.; Colón, J. L. Direct ion exchange of tris(2,2′bipyridine)ruthenium(II) into an α-zirconium phosphate framework. Inorg. Chem. 2003, 42, 2830−2832.

(43) Kumar, C. V.; McLendon, G. L. Nanoencapsulation of cytochrome c and horseradish peroxidase at the galleries of αzirconium phosphate. Chem. Mater. 1997, 9, 863−870. (44) Duff, M. R.; Kumar, C. V. Enzyme−solid interactions: important role of solvent, ions, temperature and buffer in enzyme binding to αZr(IV) phosphate. Langmuir 2009, 25, 12635−12643. (45) Nakanishi, K.; Sakiyama, T.; Kumada, Y.; Imamura, K.; Imanaka, H. Recent advances in controlled immobilization of enzymes onto the surface of the solid subst and its possible application to proteomics. Curr. Proteomics 2008, 5, 161−175. (46) Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of enzyme secondary structures. Acta Biochim. Biophys. Sinica 2007, 39, 549−559. (47) Johnson, W. C., Jr. Protein secondary structure and circular dichroism: a practical guide. Proteins: Struct., Funct., Genet. 1990, 7, 205−214. (48) Díaz, A.; David, A.; Pérez, R.; González, M. L.; Báez, A.; Wark, S. E.; Zhang, P.; Clearfield, A.; Colón, J. L. Nanoencapsulation of insulin into zirconium phosphate for oral delivery applications. Biomacromolecules 2010, 11, 2465−2470. (49) Akkuş Ç etinus, Ş.; Nursevin Ö ztop, H. Immobilization of catalase into chemically crosslinked chitosan beads. Enzyme Microb. Technol. 2003, 32, 889−894. (50) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enz. Microb. Technol. 2007, 40, 1451−1463. (51) Bhambhani, A.; Kumar, C. V. Enzyme−inorganic nanoporous materials: differential scanning calorimetric studies and enzyme stability. Microporous Mesoporous Mater. 2008, 109, 223−232. (52) Norde, W. Protein adsorption at solid surfaces: a thermodynamic approach. Pure Appl. Chem. 1994, 66, 491−49. (53) Duff, M. R.; Kumar, C. V. Molecular signatures of enzyme−solid interactions: thermodynamics of enzyme binding to α-Zr(IV) phosphate nanoplates. J. Phys. Chem. B 2009, 113, 15083−15089. (54) Loladze, V. V.; Ermolenko, D. N.; Makhatadze, G. I. Heat capacity changes upon burial of polar and nonpolar groups in enzymes. Enzyme Sci. 2001, 10, 1343−1352. (55) Pattammattel, A.; Deshapriya, I. K.; Chowdhury, R.; Kumar, C. V. Metal-enzyme frameworks: role of metal ions in promoting enzyme self-assembly on α-zirconium(IV) phosphate nanoplates. Langmuir 2013, 29, 2971−2981. (56) Grant, K. B.; Kassai, M. Major advances in the hydrolysis of peptides and proteins by metal ions and complexes. Curr. Org. Chem. 2006, 10, 1035−1049. (57) Hartwig, A. Zinc finger proteins as potential targets for toxic metal ions: differential effects on structure and function. Antioxid. Redox Signaling 2001, 3, 625−34. (58) Bhambhani, A.; Kumar, C. V. Enzyme/DNA/inorganic materials: DNA binding to layered α-zirconium phosphate enhances bound enzyme structure and activity. Adv. Mater. 2006, 18, 939−942. (59) Chowdhury, R.; Stromer, B.; Pokharel, B.; Kumar, C. V. Control of enzyme−solid interactions via chemical modification. Langmuir 2012, 28, 11881−11889. (60) Yan, J.; Du, Y.-Z.; Chen, F.-Y.; You, J.; Yuan, H.; Hu, F.-Q. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles. Mol. Pharm. 2013, 10, 2568−2577. (61) Decher, G.; Hong, J.-D. Buildup of ultrathin multilayer films by a self-assembly process: I. Consecutive adsorption of anionic and cationic bipolar amphiphiles. Makromol. Chem., Macromol. Symp. 1991, 46, 321−327. (62) Zoldak, G.; Zubrik, A.; Musatov, A.; Stupak, M.; Sedlak, E. Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure. J. Biol. Chem. 2004, 279, 47601−47609. (63) Mudhivarthi, V. K.; Cole, K. S.; Novak, M. J.; Kipphut, W.; Deshapriya, I. K.; Zhou, Y.; Kasi, R. M.; Kumar, C. V. Ultra-stable 14015

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hemoglobin-poly(acrylic acid) conjugates. J. Mater. Chem. 2012, 22, 20423−20433. (64) Thilakarathne, V.; Briand, V. A.; Zhou, Y.; Kasi, R. M.; Kumar, C. V. Enzyme polymer conjugates: improving the stability of hemoglobin with poly(acrylic acid). Langmuir 2011, 27, 7663−7671.

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