Novel biohybrids of layered double hydroxide and lactate

Journal of Molecular Structure 1105 (2016) 381e388

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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies Mohamed Amine Djebbi a, b, *, Mohamed Braiek b, Slah Hidouri a, Philippe Namour b, c, Nicole Jaffrezic-Renault b, Abdesslem Ben Haj Amara a a Laboratoire de Physique des Mat eriaux Lamellaires et Nano-Mat eriaux Hybrides (PMLNMH), Facult e des Sciences de Bizerte, Universit e de Carthage, Zarzouna, 7021, Tunisia b Institut des Sciences Analytiques UMR CNRS 5280, Universit e Claude Bernard-Lyon 1, 5 Rue de la Doua, 69100, Villeurbanne, France c Irstea, MALY, 5 Rue de la Doua, 69100, Villeurbanne, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 April 2015 Received in revised form 30 September 2015 Accepted 19 October 2015 Available online 26 October 2015

The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ionexchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UVeVisible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process. © 2015 Elsevier B.V. All rights reserved.

Keywords: Bioinorganic system Layered double hydroxide (LDH) Lactate dehydrogenase (Lac Deh) Enzyme immobilization Catalytic activity

1. Introduction Bioinorganic systems are nanostructured biohybrid materials in which a biomolecule is assembled to nanosized inorganic solid [1]. They constitute a new generation of materials, at the interface of biology and materials science [2]. These materials have been subjected to intense research not only as ecological materials, but also for other applications including biotechnology such as in biosensor systems [3,4]. Therefore, immobilization of biomolecules, such as DNA, ATP, nucleosides and enzyme with isoelectric point varying in

riaux Lamellaires et * Corresponding author. Laboratoire de Physique des Mate riaux Hybrides (PMLNMH), Faculte des Sciences de Bizerte, Universite Nano-Mate de Carthage, Zarzouna, 7021, Tunisia. E-mail addresses: [email protected], [email protected] (M.A. Djebbi). http://dx.doi.org/10.1016/j.molstruc.2015.10.065 0022-2860/© 2015 Elsevier B.V. All rights reserved.

a large pH domain has been extensively studied [5,6]. Bioinorganic research on biohybrids can protect the biomolecules from decomposition or denaturation, which made it useful in safe and targeted. 2D-Layered mineral materials, such as layered double hydroxide (LDH), so called anionic clays or hydrotalcites (HTs), has attracted considerable attention as host structures due to its technological importance in catalysis, separation technology, optics, nanocomposites materials engineering, and medical science [7]. More recently these inorganic materials have been also applied in immobilization of enzyme for biosensor development [8,9]. LDH are synthetic lamellar solids with positively charged brucite-like layers of mixed metal hydroxides separated by interlamellar domains occupied by anions and water molecules, defined by the general formula [M2þ1-xM3þx(OH)2]xþ[(A x/n)n.yH2O] (abbreviated as M2þM3þeA, where M2þ is a bivalent cation (such as Mg2þ, Ni2þ, Ca2þ, Mn2þ, Co2þ or Zn2þ), M3þ is a trivalent cation (such as Al3þ,

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Cr3þ, Ga3þ or Fe3þ), A is an interlayer anion (such as CO3 2 , SO4 2 , Cl or NO3 ) or an organic species, x represents the molar ratio [M2þ/(M2þþM3þ)] and y is the number of water molecules located in the interlayer region together with anions) (Scheme 1). These compounds, due to its layered structure display a remarkable range of physic-chemical properties make them an attractive choice to immobilize enzymes: (i) adjustable chemical composition layer/ interlayer; (ii) variable anionic exchange capacities 1.5e4.5 meq/g; (iii) opened structure which can accommodate large anionic molecules; (iv) and poor adjustable textural properties controlled by the synthesis process and conditions. Anions such as enzymes are typically immobilized into LDH by three approaches [10,11]. The first approach is the co-precipitation method, which requires the addition of a solution of M2þ and M3þ ions into a base solution of the desired anions. The second technique is the direct exchange method, where LDH is stirred in a solution of the chosen anions at a suitable concentration. The last method is the rehydration method, where the calcined LDH is added to a solution of desired anions [12]. Several studies showed that the LDHs are a good support for enzyme immobilization, due to their easy synthesis procedure combined with good retention capacity of enzymes [13]. Moreover, they can play a key role not only as matrices for enzyme support, but also to preserve their activity and their interesting properties in charge transport. However, lactate dehydrogenase (Lac Deh) enzyme is chosen as a model enzyme, being immobilized in LDH materials. Lac Deh is an enzyme which largely presented in the organs and tissues of vegetal or animal organizations (kidney, heart, muscles, pancreas, skin …) [14], catalyzes the conversion of pyruvate to lactate and back, as it converts NADH to NADþ and back. Measurement of lactate using biosensors is of great importance for the clinical analysis as well as for food analysis [15]. For these reasons, several types of enzyme support have been reported in literature [16e20] to meet primarily the need for the development of continuous monitoring techniques. Despite the large number of matrices used as support, no paper concerning the immobilization study into an anionic clay matrix, belonging to the LDH class, has been reported so far. In the present investigation, we attempted to immobilize Lac Deh on two types of hydrotalcites, MgAl and ZnAl LDH materials via ion-exchange and co-precipitation process. In addition, we systematically studied the merits and demerits of these immobilization methods. 2. Experimental 2.1. Starting materials Lactate Dehydrogenase (EC 1.1.1.27, molecular weight 140 kD) was purchased from Biomagreb. b-nicotinamide adenine dinucleotide (NAD), MgCl2.6H2O, ZnCl2, AlCl36H2O and NaOH were purchased from Sigma Aldrich. Phosphate-buffered saline (PBS), and distilled water was used during all experiments.

2.2. Synthesis of MgAleCl and ZnAleCl LDHs The MgAleCl and ZnAleCl LDH hybrid inorganic materials were prepared by the co-precipitation route. Typically, 50 mL of the magnesium salts MgCl2 or zinc salts ZnCl2 and aluminum salts AlCl3 were prepared in total cationic concentration of 1.0 M. In order to keep a constant M(II)/Al ratio of 2, the amount of M(II)Cl2 and AlCl3 was adjusted for each synthesis. The salt solution was then added drop wise into a reactor with a constant flow of 0.12 mL/min. Throughout this addition, the pH of the solution was then elevated and maintained using 2.0 M NaOH solution, which caused metal coprecipitation until the solution reached a pH of 9 and 7.5 respectively for MgAl and ZnAl phases. The reaction was carried out under N2 atmosphere to avoid carbonate contamination. The addition of the salt solution was complete within 7 h and the suspensions were immediately centrifuged at 5000 rpm without any ageing in order to quench the crystal growth and therefore obtain small platelets. The solids recovered by centrifugation were washed several times with distilled water until no sodium chloride was present and dried at room temperature for 24 h. 2.3. Adsorption studies Adsorption of Lac Deh into the MgAleCl and ZnAleCl LDHs was carried out by direct ion-exchange reaction. Briefly, 5 mL of Lac Deh solutions at different concentrations (Ci) were prepared in a phosphate buffer saline solution (0.1 M and pH ¼ 7.4). Next, these solutions were slowly added to a suspension containing 5 mg of a freshly prepared LDH in 5 mL of distilled water. This system was maintained under magnetic stirring at 25 C for 2 h. Afterwards, the solid product was isolated by centrifugation, washed thoroughly with distilled water and dried overnight at room temperature. The resulting biohybrid material was denoted LDH/Lac Dehads. The amount of Lac Deh non-immobilized and present in water of washing was determined by absorbance UVeVis at 340 nm. 2.4. Synthesis of MgAleCl/Lac Deh biohybrid via co-precipitation The co-precipitation method described above was adapted for the preparation of small amounts of biohybrid materials. Typically, 10 mL of a 0.1 M metallic salts solution (mixed aqueous solution of MgCl2 and AlCl3 with a molar ratio R ¼ Mg2þ:Al3þ of 2:1) was introduced with a constant flow into reactor containing an aqueous Lac Deh solution under stream of N2. The pH was maintained constant at 9 by simultaneous addition of a 0.2 M NaOH solution. The co-precipitation was performed for LDH/Lac Deh mass ratio Q equal to 1.0, 98% of the enzyme is immobilized. The resulting material was collected and stored following the same procedure as described above and denoted MgAleCl/Lac Dehcop. 2.5. Determination of enzyme activity and kinetics parameters Activity measurements and kinetic study of free and immobilized lactate dehydrogenase were measured in the direction of oxidation of lactate to pyruvate (Eq. (1)) by monitoring the changes in the absorbance of NADH at 340 nm according to the method described by Sohn et al. [20]. Lac Deh

Lactate þ NADþ ! Pyruvate þ NADH þ Нþ

Scheme 1. Schematic representation of the crystal structure of the layered double hydroxide.

(1)

Typically, 2 mL of 0.1 mM NAD solution and 3 mL of 1.0 mM lactate solution were added to a test tube containing free enzyme or immobilized enzyme (MgAleCl/Lac Deh and ZnAleCl/Lac Deh). All assays were conducted at 25 C in PBS buffer medium (pH ¼ 7.4). After the reaction mixture was stirred shortly by vortexing the


383

increase in NADH absorbance was measured spectrophotometrically at 340 nm for 10 min. The immobilized Lac Deh activity yield was calculated in terms of relative immobilized Lac Deh activity on LDH material as a percentage using the difference in Lac Deh activity between free and immobilized forms. Kinetic constants (Km and Vmax) values were also determined; the reaction of lactate and NAD catalyzed by Lac Deh was also performed in a cuvette containing free enzyme or 1 mg of immobilized enzyme. The test solution required the addition of 70 mL of lactate and 200 mL of NAD to 730 mL PBS. Finally, the rate of absorbance increase of the formed NADH was measured at 340 nm as a function of time. The maximum enzymatic reaction rates (Vmax) and MichaeliseMenten constants (Km) for Lac Deh were calculated from the data using a LineweavereBurk plot. 2.6. Instruments Several physical-chemical techniques were employed in the characterization of the obtained materials: Powder X-ray diffraction patterns (XRD) were collected with a conventional Bragg-Brentano geometry (q - 2q scans) on a Bruker D8 Advance automated diffractometer using CuKa radiation (l ¼ 1.54060 Å) at 40 kV and 30 mA, and continued scanning mode. The patterns were recorded from 2 to 70 2q in steps of 0.02 2q, with the measuring time of 86.79 s per step. Fourier transform infrared spectra (FTIR) in ATR mode were recorded using a Nicolet 200 FTeIR (Thermo Scientific) spectrophotometer in the range of 4000e400 cm-1. Quantitative determination of immobilized enzyme and enzyme activity measurements were acquired by UVeVisible spectroscopy using Shimadzu UVmini-1240 UVeVisible spectrophotometer. The dispersion of the enzyme within the LDH layers was controlled by transmission electron microscopy (TEM). The TEM images were obtained from a Tecnai G2 using an accelerator voltage of 200 kV. The bioinorganic components appeared black/gray on the micrographs.

Fig. 1. Adsorption isotherms of Lac Deh onto MgAleCl and ZnAleCl LDHs.

where Cs is the adsorbed concentration (mg/mg), Ce is the equilibrium concentration (mg/mL), Cm and Kf are constants that estimate the adsorption capacity according to Langmuir and Freundlich respectively, while L and n are constants that estimate the affinity of the adsorbate-adsorbent according to Langmuir and Freundlich respectively. The calculated parameters of the Langmuir and Freundlich isotherms are listed in Table 1. The correlation coefficients R in Table 1 indicate that the sorption isotherms for Lac Deh fitted well to the Langmuir model, and the equilibrium adsorption capacity determined by the Langmuir model is consistent with the experimental results. This indicates that enzyme adsorption onto LDHs layers is characterized by occupying all the available active sites as well as the LDHs surface edges [22,23]. This fact confirms that the adsorption process occur in a monolayer adsorption mechanism. 3.2. Characterization

3. Results and discussion 3.1. Adsorption isotherms With an isoelectric point of 6.5, Lac Deh is negatively charged at pH 7 and consequently it can be favorably adsorbed on LDH particles by electrostatic attractions. The affinity between Lac Deh and LDH matrices has been investigated by the adsorption method. Fig. 1 shows the adsorption isotherms of Lac Deh on MgAleCl and ZnAleCl phases. Indeed, they cannot be considered clearly as pure L-type, according to the classification of Giles et al. [21]. One can distinguish two different parts on these adsorption isotherms. At low equilibrium concentration Ce (below 0.3 mg/mL), a net increase of the amount of Lac Deh adsorbed is observed and beyond this value one observes a plateau related to the capacity of adsorption at 0.41 and 0.44 mg/mg, respectively, for MgAleCl and ZnAleCl phases. These results suggest that the adsorption proceeds in two steps: linear progression of the adsorption followed by saturation of the external sites of the mineral structure by the enzyme. Several models were developed to describe adsorption phenomena. Here, the sorption data was analyzed according to Langmuir (Eq. (2)) and Freundlich (Eq. (3)) equations (Fig. 2): Ce/Cs ¼ Ce/Cm þ 1/ (Cm*L)

(2)

log Cs ¼ log Kf þ (1/n)*log Ce

(3)

3.2.1. X-ray diffraction Two different LDH matrices MgAleCl and ZnAleCl were prepared for the immobilization of Lac Deh by the adsorption process. In order to show their possible protective feature as host structure, X-ray diffraction investigations (Fig. 3) were carried out. The XRD patterns (Panel (A) and (B)) display the characteristic diffraction peaks (003), (006), (012) and (110) of pure LDH compounds. These XRD peaks have been indexed assuming a hexagonal lattice with a R3m symmetry. Cell parameters c and a of the Rhombohedral structure were determined from the positions of the (003), (006) and (110) diffraction peaks, respectively. The lattice parameter a ¼ 2d(110) corresponds to an average cationecation distance calculated from the (110) reflection, while the c parameter corresponds to three times the thickness of d003 parameter. In this case c was calculated from two diffraction lines using equation c ¼ 3/2 [d003þ2d006] [24]. Determined cell parameters c and a are shown in Table 2. The XRD patterns of MgAleCl and ZnAleCl LDH/Lac Deh biohybrids (LDH/Lac Deh mass ratio of 1.0) prepared using ion exchange methods are given in Panel (A) and (B). The (00l) peaks remain unchanged under the immobilization of Lac Deh indicating that any intercalation of the enzyme is never obtained as for alkaline phosphatase [25], trypsin [26], urease [27] in the LDH layers. However, XRD patterns of as-prepared biohybrids show little changes compared to the diffractograms of reference materials, manifested by reduction in the intensity of diffraction line.

384


Fig. 2. Modeling according to the equations of Freundlich and Langmuir.

Therefore one can say that this synthesis method does not affect the crystallographic coherence domain of LDH structure and one conclude that the immobilization of Lac Deh is established on the surface of the LDH support. The co-precipitation route of Lac Deh was only used for MgAleCl hybrid phase because this enzyme has an optimum pH between 8.8 and 9.8 and the basic nature of MgAleCl material could stabilize the enzyme under operating conditions (to prevent the denaturation of the enzyme and to adapt the pH in optimum activity). The spatial orientation of the co-precipitated phase is represented in Panel (A). A strong decrease of the diffraction lines intensities and an enlargement of the diffraction lines compared to the reference diffractogram, confirm both a reduction of the particle size according to the DebyeeScherrer equation (Table 2), and a greater disorder in the structure. These structural changes arise from a better dispersion of the inorganic layers in the bioorganic matter. The preservation of the (012) and (110) diffraction lines prove

the formation of the layer structure. The (00l) lines remain unchanged (not shifted to lower 2q angles), reveal no expansion of the interlamellar distance and indicate that the immobilization process does not lead to the intercalation of Lac Deh within the LDH layers. 3.2.2. FT-IR analysis FTeIR is often used to identify the functional groups and chemical bonds present in compound because there is a specific absorption and wavenumber for each functional group. Therefore, this technique can be used to complement other techniques, confirming that the immobilization has occurred instead of the intercalation. FTeIR analysis of the different biohybrids phases are shown in Fig. 4 (Panel (A) and (B)), confirm the presence of both the enzyme and the inorganic network. They show four characteristic absorption bands around 2900, 1550, 1100 and 950 cm1 related to the enzyme, which could be attributed to the amide groups (eNH2), the

Table 1 Langmuir and Freundlich parameters for Lac Deh adsorption on MgAleCl and ZnAleCl LDHs. LDH/Lac Deh

MgAleCl/Lac Dehads ZnAleCl/Lac Dehads

Cs (mg/mg)

Langmuir Cm (mg/mg)

L (mL/mg)

R

Freundlich Kf (mg/mg)

n

R

0,41 0,44

0.5347 0.5952

6.2340 6.0003

0.9860 0.9883

0.5933 0.7189

1.7796 1.8518

0.9748 0.9530


385

Fig. 3. XRD patterns for MgAleCl/Lac Deh (A) and ZnAleCl/Lac Deh (B) prepared using the two different methods.

Table 2 Properties of LDH/Lac Deh prepared using the two different methods. Properties

LDH compound MgAleCl

Basal spacing, nm d110, nm Lattice parameter a, nm Lattice parameter c, nm Crystallite size* in c direction, nm

0.7803 0.1525 0.3050 2.3375 21.4

LDH/Lac Deh compounds ZnAleCl

0.7825 0.1539 0.3078 2.3416 32.6

Via ion-exchange

Via co-precipitation

MgAleCl/Lac Dehads

ZnAleCl/Lac Dehads

MgAleCl/Lac Dehcop

0.7692 0.1520 0.3040 2.307 14.0

0.7922 0.1549 0.3098 2.3766 27.8

0.7712 0.1526 0.3052 2.3136 6.8

*

Debye-Scherrer equation: D ¼ kl/bcosq (where k denotes the Scherrer's constant (k ¼ 0.9), l is the wavelength of the radiation used, b the full-width at half-maximum of diffraction line and q is the Bragg diffraction angle).

aromatic groups (eCs¼Ce), CeC or CeN bending and CeH out-ofplane bending of aromatic groups of enzyme structure [28e30], respectively. There is no difference observed between the signature of free and immobilized Lac Deh. They are identical in numbers and positions which confirms that immobilization of Lac Deh in LDH

materials does not affect the molecular structure of the enzyme. The vibration bands of LDH phases are also unaffected: a broad absorption band at 3400 cm1 corresponds to the OeH hydroxyl group that is present within the layer. The band recorded at 1360 cm1 is associated with the symmetric vibration of the

Fig. 4. FTeIR spectra of MgAleCl/Lac Deh (A) and ZnAleCl/Lac Deh (B) prepared using the two different methods.

386


anionic carbonate functions ðCO3 2 Þ of interlayer Cl anions. The bending vibration of H2O appears at 1640 cm1 while the bands observed in the low frequency region of the spectrum at 850 cm1 to 600 cm1 and 440 cm1are interpreted as the lattice vibration modes and can be assigned to the MO and OeMeO stretching modes. The increase of the immobilized Lac Deh amount (Q) attributed using the two different methods is shown by the increase of the intensities of the Lac Deh vibrations bands relatively to the lattice vibrations. Moreover, the band at 1100 cm1 is more intense for the biohybrid materials prepared by adsorption suggesting the presence of the phosphate ions coming from the buffer phosphate used to disperse the Lac Deh and that the binding adsorption mechanism might be somewhat different than the co-precipitation mechanism. The enlargement and the decrease of the intensity of the OeH stretching vibrations for the biohybrid phases compared to the reference phases, reveals the existence of hydrogen bond network connecting enzyme molecules and matrix. The immobilization of Lac Deh onto the LDH layers is done by retaining a high rate of hydration that allow saving their structural integrity. 3.2.3. Transmission electron microscopy To determine the effect of the immobilization process of Lac Deh on the LDH morphology, the surface morphology of LDH before and after immobilization was observed. Fig. 5 present TEM images of MgAleCl LDH and biohybrids MgAleCl/Lac Deh. The nanograph of MgAleCl precursor (Panel (A)) indicates distinct individual particles of hexagonal shape and a size smaller than 200 nm which could result in the formation of clusters. When Lac Deh enzyme is immobilized in the MgAleCl phase, TEM confirms that a slight modification of the aggregation is obtained during the adsorption process due to the presence of enzyme molecules face to face with the LDH platelets aggregates (Panel (B)) while in the case of the coprecipitated process (Panel (C)), a dense aggregation of biohybrid occurs. Indeed, the adsorption mechanism appears as a partial process of anionic exchange on the surface of LDH crystallites, whereas the presence of the enzyme in the precipitation medium is opposed to the crystalline growth of LDHs. LDH Particles seem to be dispersed in an enzyme film that makes us think about strong interaction and association between Lac Deh molecules and LDH particles.

was set to 100% as a reference. The immobilization process shows an activity lower than the free enzyme. This means that there is a partial loss of Lactate Dehydrogenase activity during the chemical reaction in the immobilization process. Previous works [16e18] on oriented immobilization of Lactate Dehydrogenase have been explored in literature indicating a hindered diffusion of the substrate that leads to a partial lower substrate concentration after immobilization process than that in solution. However, Lactate Dehydrogenase immobilized via ion-exchange shows the highest activity around 75% on MgAleCl LDH and 78% on ZnAleCl LDH than those immobilized via co-precipitation method which is around 44% on MgAleCl LDH. Unlike the co-precipitation route which suggests a strong/high adsorption of the substrate, the preceding findings can be accounted for by the accessibility of the substrate to the active site of the enzyme molecules in the adsorption process. Similarly, in their work, Mansouri et al. [26], showed that the low activity was attributed to a co-precipitation method. 3.3.2. Kinetics response To further understand the loss of activity after immobilization, we analyzed the change of the kinetic constants of Lactate Dehydrogenase before and after immobilization. LineweavereBurk plot over substrate (NAD) concentration range of 0.5e4 mM for both the free and immobilized enzyme at 25 C obeys the MichaeliseMenten law of enzyme kinetics. Table 3 allows comparisons of immobilized and free enzyme as a catalyst for the lactateepyruvate conversion. Usually, apparent Km values of immobilized enzymes are higher, and Vmax values are lower than those for free enzyme, which is consistent with earlier reported works [16,17,30e32]. However, the higher value of Km of the immobilized enzyme is most likely caused by the partial structural change of the active sites for the binding of substrate after immobilization due to diffusion limitations and steric hindrances in the immobilized forms. The decrease in Vmax of the immobilized LDH might be the result of the change in the catalytic efficiency of the immobilized LDH. The difference of Km and Vmax between different formulations prepared via ion-exchange and co-precipitation can be attributed to the limited accessibility of substrate molecules to the active sites of the immobilized enzyme, as a result of the spatial distribution of the enzyme molecules in the LDH layer and the partial conformational changes of enzyme molecules caused by Van Der Walls and homophilic interactions.

3.3. Activity study 3.3.1. Activity of the lactate dehydrogenase-immobilized LDH Fig. 6 shows the relative activity of free and immobilized enzyme. The enzyme activity of the first run for the free enzyme

3.4. Correlation among structure/microstructure and enzyme activity In light of these results, one can try to establish a correlation

Fig. 5. TEM images of MgAleCl (A), MgAleCl/Lac Dehads (B) and MgAleCl/Lac Dehcop (C) samples.


RelaƟve enzyme acƟvity (%)

120

387

Lac Deh

100

ZnAl–Cl/Lac Deh MgAl–Cl/Lac Deh

80

Free enzyme immobilized enzyme via ion-exchange Immobilized enzyme via coprecipitaƟon

60 40 20 0

Biohybrid LDH/Lac Deh phases Fig. 6. Comparison of the relative activity of free and immobilized enzyme.

Table 3 Kinetic parameters for the free and immobilized enzyme. Formulation

Km (mM)

Vmax (mM/min/mg)

Free Lactate Dehydrogenase MgAleCl/Lac Deh (adsorption) ZnAleCl/Lac Deh (adsorption) MgAleCl/Lac Deh (co-precipitation)

3.300 5.989 5.889 4.864

29.81 3.75 3.73 0.79

between the structure/microstructure and the enzymatic performance of lactate dehydrogenase after immobilization in LDH matrix. A high content of enzyme on the target surface does not truly indicate the success of the immobilization process. It is also necessary to determine enzymatic activity, which is the main function of immobilized biomolecules in an enzyme based biosensor. The influence of the association between lactate dehydrogenase and LDH layers on the catalytic activity can be assessed by comparing structure/microstructure of adsorption and coprecipitation mechanism. The structural and morphological study shows that the co-precipitation mechanism affects the crystallinity of LDH phase with the creation of stacking fault defects and a structural distortion on the layers. The link created during the coprecipitation decrease the catalytic activity and causes a change in the conformation of the enzyme and/or steric hindered. On the contrary, in the adsorption mechanism the enzyme feels freer to create comfortable links for its catalytic activity. Scheme 2 displays a realistic model of the association between lactate dehydrogenase and LDH hybrid materials. 4. Conclusions New bioinorganic materials were synthesized by immobilizing lactate dehydrogenase (Lac Deh) onto MgAleCl and ZnAleCl layered double hydroxide (LDH) compounds using ion-exchange and co-precipitation techniques. The role of the metal cations of LDH layers on the LDHeenzyme interactions and consequently on the enzyme preservation activity was investigated. The enzyme-

Scheme 2. Ideal representation of the LDHeLactate Dehydrogenase interactions.

immobilized LDH particles were characterized using XRD, FT-IR and TEM. From the XRD patterns, it was inferred that the immobilization process does not lead to the intercalation of the enzyme within the LDH layers. The FT-IR spectra confirmed the immobilization of Lac Deh onto the LDH particles, but suggested that their binding mechanisms via the two processes might be somewhat different. The nanographs analysis displays two models of the hybrid materials structure in which non-exfoliated (using adsorption) and partial exfoliated (using co-precipitation) layers coexist in interactions with lactate dehydrogenase. The immobilization efficiency was also influenced by the immobilization process; however, immobilized amount of enzyme in inorganic matrix was estimated out for the first time and catalytic activity was determined for the second time. These preliminarily results suggest that the use of LDH host structure for lactate dehydrogenase immobilization is compatible with the preservation of the enzyme activity and showed the potential of using biohybrid for bioprocess monitoring systems as biosensors or biofuel cells. Using the immobilized Lac Deh onto LDH materials, we are currently working on the fabrication of bio-electrode layered double hydroxide-lactate dehydrogenase for construction of lactate/O2 biofuel cell. Notes The authors declare no competing financial interest. Acknowledgment I would like to thank graciously Aisha Gharsalli from LECOM School of Dental Medicine, FL 34211, Etats-Unis, for her constructive discussions and comments on an earlier version of this article. References [1] L. He, C.-S. Toh, Anal. Chim. Acta 556 (2006) 1e15. [2] W. Jin, J.D. Brennan, Anal. Chim. Acta 461 (2002) 1e36. [3] J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeong, J. Portier, J. Am. Chem. Soc. 121 (1999) 1399e1400. [4] J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeong, J. Mater. Chem. 11 (2001) 1671e1674. [5] J.H. Choy, J.S. Jung, J.M. Oh, M. Park, J.Y. Jeong, Y.K. Kang, O.J. Han, Biomaterials 25 (2004) 3059e3064. [6] J.V. de Melo, S. Cosnier, C. Mousty, C. Martelet, N. Jaffrezic-Renault, Anal. Chem. 74 (2002) 4037e4043. [7] D. Shan, S. Cosnier, C. Mousty, Biosens. Bioelectron. 20 (2004) 390e396. [8] C. Mousty, L. Vieille, S. Cosnier, Biosens. Bioelectron. 22 (2007) 1733e1738. [9] D. Shan, S. Cosnier, C. Mousty, Anal. Chem. 75 (2003) 3872e3879. [10] P.S. Braterman, Z.P. Xu, F. Yarberry, in: S.M. Auerbach, K.A. Carrado, P.K. Dutta (Eds.), Marcel Dekker, Inc. New York 2004 373e474. [11] C. Forano, T. Hibino, F. Leroux, C. Taviot-Gueho, in: F. Bergaya, B.K.G. Theng, G. Lagaly (Eds.), Elsevier New York 2006 1021e1095. [12] H.W. Olfs, L.O. Torres-Dorante, R. Eckelt, H. Kosslick, Appl. Clay Sci. 43 (2009) 459e464. vot, in: E. Ruiz-Hitzky, K. Ariga, Y. Lvov, Wiley-VCH Weinheim [13] C. Forano, V. Pre 2008, 443.

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