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Protein oligomerization based on Brønsted acid reaction Andressa R. Pereira, Roberto A.S. Luz, Filipe C. D. A. Lima, and Frank Nelson Crespilho ACS Catal., Just Accepted Manuscript • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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Protein oligomerization based on Brønsted acid reaction Andressa R. Pereira§, Roberto A. S. Luz§, Filipe C. D. A. Limaǂ, Frank N. Crespilho*§ §

ǂ

São Carlos Institute of Chemistry, University of São Paulo, 13560-970, Brazil

Federal Institute of Education, Science and Technology of São Paulo, Campus Matão, 15991-

502, Brazil ABSTRACT: Oligomeric proteins are abundant in nature. However, their synthesis remains a significant challenge. Controlled oligomerization process can provide important insights into the evolution of modern proteins and the development of more efficient biocatalysts. Here, we propose a pathway for producing glucose oxidase (GOx) homooligomer (Ol-GOx), a redox enzyme with extensive biotechnological applications. We obtained Ol-GOx from the one-pot reaction of the native protein with a Brønsted acid, trifluoromethanesulfonic acid (TFMS). OlGOx had a hydrodynamic radius of 96 nm and molecular mass of 2 MDa. Ol-GOx exhibited higher thermal stability compared to the native protein, as well as increased hydrophobicity, which is a primary characteristic needed for its stabilization in the solid state for applications in bioenergy, heterogeneous catalysis, and biomedicine. Furthermore, there were remarkable improvements in redox properties and protein stabilization, suggesting that this approach for OlGOx synthesis could be used to design more efficient oligomer biocatalysts.

KEYWORDS: glucose oxidase, oligomerization, aggregation, protein–protein interaction, direct electron transfer, bioelectrocatalysis.

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INTRODUCTION A comprehensive knowledge regarding the mechanisms of protein-protein interactions is fundamental and essential in several research aspects at the interface of biochemistry, protein engineering, and cell biology. In particular, evolutionary mechanisms involved in the proteinspecific binding of homooligomers and their unique functional roles are considered important in understanding specific cellular functions. Specifically understanding biomolecular interactions can be beneficial for the identification or synthesis novel oligo-enzymes for drug development and large-scale manufacturing of bioactive molecules. Protein oligomerization involves dynamic interactions between two or more polypeptide chains.1 Oligomeric interfaces offer electrostatic and geometrical shape complementarity, increasing the specificity of the interaction.2,3 Oligomerization can also confer functional advantages to proteins, such as allosteric regulation, minimize synthesizing errors as compared to single-chain proteins, and enhance resistance to denaturation and degradation in larger proteins.4,5 Protein oligomerization can be considered as a type of protein aggregation, which depends on several intrinsic and extrinsic factors associated with protein structure levels and the protein environment, respectively.6 Protein aggregation can occur via different pathways, such as through unfolding intermediates and unfolded states, through self-association, through direct chemical linkages, and through physical aggregation process. Chemical aggregation7-9, for instance, may be used in order to improve enzyme loading and its stability. Studies suggest that the small population of folding/unfolding intermediates function as the precursor to this aggregation: the intermediates expose hydrophobic chains, which have more flexibility when compared to the native state and can facilitate the process of aggregation.6,10 Environmental


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conditions, such as pH can influence protein aggregation by affecting charge density on the protein surface and determine the predominant shape of the agglomerates. Thus, pH and hydrophobicity are considered important parameters in determining protein aggregation rates.6 In this study, we have proposed a chemical route for producing protein–protein oligomers from the corresponding native protein forms. We used native dimeric glucose oxidase (GOx, EC 1.1.3.4) to produce its oligomer (Ol-GOx) via protein reaction with a Brønsted acid. GOx is a redox enzyme with a high stability and high specificity for glucose, and was first discovered by Muller in 1928.11 It has been isolated from several sources,12-15 including Penicillium amagasakiense, citrus fruits, red algae, and the fungus Aspergillus niger, GOx has been extensively investigated for several industrial and health-based diagnostic applications. For instance, GOx is widely used as a biocatalyst in biosensors16 used for monitoring diabetes and in the quantitative determination of

D-glucose

in foodstuff, beverages, and in fermentation

processes. GOx has also been used to remove oxygen from food, improving its colour, flavour, and shelf life. Here, we propose a versatile method for GOx oligomerization based on protein reaction with a Brønsted acid, resulting in the synthesis Ol-GOx (having a molecular mass of 2 MDa) with unique activity. In addition, our results corroborate that large proteins are more resistant to degradation and denaturation. This novel approach can be suitably adopted for the oligomerization of several classes of enzymes and has valuable potential for the artificial synthesis of oligomeric biocatalysts.


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METHODS Enzyme oligomer synthesis and purification Native GOx from Aspergillus niger was incubated in the presence of TFMS at pH 1.0, 4 °C, for 30 minutes. For this, 2.0 mL of GOx solution (3.0 mg mL-1 previously prepared in 0.10 mol L-1 sodium phosphate buffer; pH 7.5) was mixed with 140 μL of TFMS. The temperature was maintained at 20 °C. Ol-GOx mixture was placed in a cellulose membrane dialysis tubing (14 kDa MWCO) to remove the residual acid and to purify the sample. Two dialysis procedures were performed. In the first dialysis, the Ol-GOx mixture with 40 mL of sodium phosphate buffer (0.10 mol L-1; pH 7.5) was shaken for 3 h at 25 ºC. In the second dialysis, the enzyme oligomer mixture with another 40 mL of sodium phosphate buffer (0.10 mol L-1; pH 7.5) was incubated at 4 °C, without shaking, for 3 h. Subsequently, Ol-GOx was removed from the cellulose membrane dialysis tubing and stored at 4 °C until further use. To compare the structure of native GOx and Ol-GOx, the following techniques were utilized: UV-VIS spectroscopy, CD and FTIR. The conditions of the measurements and the equipment that were used are described in detail in the Supporting Information. DLS experiments were performed on a Zetasizer Nano Series Malvern at 25 ºC. Native GOx and Ol-GOx were first filtered (using membranes of pore size 0.2 µm) in order to remove any impurities before the DLS experiments. The solutions were diluted to 0.5 mg mL-1 in sodium phosphate buffer (0.1 mol L-1, pH 7.5). TEM images were obtained on a JEOL JEM2100 LaB6 – 200 kV with low sample exposure time in order to analyse the size and morphological characteristics of GOx before and after the treatment with TFMS. Enzyme solutions were dialyzed in deionized water to remove any residual sodium phosphate buffer. Then, they were placed in a grid and dried overnight.


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Enzyme activity The catalytic activities of GOx and Ol-GOx were measured according to procedures described previously.17, 18 Briefly, 0.5 mL of ABTS (16 mmol L-1), 0.5 mL of peroxidase from horseradish (1.9 U mL-1) in 0.10 mol L-1 citrate-phosphate buffer solution (pH 5.2), and 0.5 mL of glucose (0.09 mol L-1) were added to a reaction tube. Reaction mixtures were purged with oxygen for 60 seconds, and then, 50 μL of GOx (3.0 mg mL-1) and Ol-GOx (3.0 mg mL-1) were added to the mixture in separate reaction tubes. The flasks were incubated for 30 minutes at 30 °C, and the reaction was stopped with 0.10 mL of 4.0 mol L-1 HCl. The product formed in the final reaction was ABTS+, which was detected using UV-VIS spectroscopy, having absorption maxima at 420 nm.

Electrochemical measurements Carbon fibers were used as working electrode, as described previously.19 The GOx and Ol-GOx-modified electrodes were prepared by the physical adsorption of the enzyme, when the fiber array was placed in the enzyme solution for 36 h at 4 °C. Next, 20 μL of 2.5 %, Nafion® solution in phosphate, pH 7.0, was dropped onto the fibers of the electrode, and the bioelectrode was vacuum-dried. Electrochemical experiments were performed using a three-electrode system. The details are provided in the Supporting Information.

RESULTS AND DISCUSSION In protein oligomerization, the pH is a key parameter for the distribution of charge on the protein surface, which affects intermolecular folding as well as protein–protein interactions.20 In this case, the aggregation depends on hydrophobic attraction and electrostatic repulsion.6,20 It has


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been reported that small and ordered aggregates show higher proportions of hydrophobic residues on their surfaces, which remain typically buried in a folded protein structure.21 Furthermore, it has been reported that hydrophobicity and the propensity for the conversion of αhelical structure to β-sheet contribute positively to aggregation rates.20 In this context, we demonstrate how the Ol-GOx oligomeric form was obtained from the native form of the protein (GOx). GOx oligomerization was performed by the destabilization of its ternary structure (by a process conventionally known as protein aggregation). Native GOx was incubated in the presence of a Brønsted acid (trifluoromethanesulfonic acid; TFMS) at pH 1.0 and 4 °C. TFMS was selected as the Brønsted acid for the synthesis of Ol-GOx oligomers as it does not degrade the protein,22 preserving the catalytic activity of the enzyme. Others Brønsted acids (e.g. sulfuric, nitric, and hydrochloric acids) were also evaluated for Ol-GOx synthesis. Although these Brønsted acids promoted enzyme aggregation in a similar manner to that of TFMS (see dynamic light scattering (DLS) measurements, Figure S1 in the Supporting Information), the resulting OlGOx enzymes were inactive. Therefore, the protein reaction with TFMS was considered efficient for Ol-GOx synthesis, as it does not degrade the protein,22 resulting in an intact monomeric unit as observed from SDS-PAGE experiments (Figure S2a in the Supporting Information). Furthermore, the Ol-GOx was able to retain it catalytic properties for glucose oxidation, as will be described later. Figure 1a shows the UV-VIS spectrum for native GOx and Ol-GOx obtained by TFMS reaction, where the increase in the turbidity for Ol-GOx aqueous suspension can be clearly observed (inset, Fig. 1a), suggesting that the drastic decrease in pH (pH 1.0) during the incubation process causes the agglomeration and enzyme oligomerization. The oligomerization reaction did not affect the prosthetic group, flavine adenine dinucleotide (FAD) (Fig. 1a), and the


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molar absorptivity (ε) remains the same (Figure S2b, c and d in the Supporting Information), which have been discussed in further detail later. UV-VIS spectrum of GOx shows three absorption bands at 276, 382, and 453 nm (Fig. 1a, red line), where the band with the highest intensity is assigned to the aromatic amino acid side chains23,24 and to the riboflavin present in prosthetic group,25 whereas the other two broad intensity bands are assigned to semiquinone groups in the oxidized state present in FAD.25 For Ol-GOx, the intensity of absorption at 276 nm was observed to the same as that of GOx (Fig. 1a, blue line), which implies that the prosthetic group FAD remained bound to enzyme after oligomerization. Additionally, the semiquinone group showed a relatively more discreet absorption associated with the increase in baseline, as a consequence of the increase in light scattering caused by the appearance of turbidity. In fact, a 3.0 mg mL-1 suspension exhibited a milk-like appearance as shown in the inset of Fig. 1a. It should be noted that turbidity cannot be used as a quantitative parameter to monitor Ol-GOx formation, as it does not quantify the absolute number of monomers in the same oligomer. Turbidity occurs because the observed light scattering will depend on both, the size and number of particles.26 On the other hand, turbidity provides the means for a visual inference: solutions that appear more opalescent to the eye show increased absorbance at 350 nm. In fact, there is a difference in transmittance exhibited by both enzymes at 350 nm—80% for native GOx and 59% for Ol-GOx—indicating the increase in turbidity after oligomerization. The increase in turbidity is associated with oligomer formation and it can be related to the changes in the hydrophobicity of the protein. Hydrophobic interactions are important in defining homo-oligomeric interfaces1 and consequently, influences the organization of water molecules around the Ol-GOx. DLS measured at pH 7.5 and 25 °C (Fig. 1c) shows a hydrodynamic diameter (hd) of 9±1 nm for a sphere-modeled native GOx,27 and hd of 96±15 nm for Ol-GOx.


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This 10-fold increase in hd suggested that the Ol-GOx is composed of 10 dimeric protein molecules, which was confirmed by transmission electron microscopy (TEM) images (Fig. 1d). Furthermore, TEM images (Figure S3 in the Supporting Information) corroborate the DLS results (Fig. 1c), showing dimeric GOx sizes and Ol-GOx aggregation.

Figure 1. Spectral and microscopic characteristics of GOx and Ol-GOx. (a) UV-VIS spectra of GOx solution (3.0 mg mL-1; red line) and Ol-GOx solution (3.0 mg mL-1; blue line); Inset: Images of solutions of GOx (left side) and Ol-GOx (right side); (b) CD spectra of GOx solution (red line) and Ol-GOx solution (blue line), both prepared in 0.10 mol L-1 sodium phosphate buffer (pH 7.5); (c) Size distribution by number obtained in DLS measurements for GOx (red line) and Ol-GOx (blue line); (d) TEM images of Ol-GOx; (e and f) Deconvoluted amide I bands of GOx and Ol-GOx, respectively, obtained using FTIR spectroscopy.

Figure 1b shows the circular dichroism (CD) spectra with the characteristic band for the α-helix structure28 at 220 nm. On deconvolution, using CDNN software,29 the results suggest that


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no significant changes occurred in the secondary structure of the native protein. Considering that two GOx molecules bind to each other when they are folded, CD was also used to determine whether there are changes in the apparent midpoint of the unfolding transition (TM) or if the slope of a transition changes when oligomers are present. The thermodynamics of folding based on the curves of ellipticity as function of temperature (in the range 20–90 °C) shows a decrease in the molar ellipticity when temperature increases (Figure S4 in the Supporting Information), which occurs due to thermal denaturation.30,

31

The TM for GOx (65 ºC) is higher than that

obtained for Ol-GOx (59 ºC); however, the shift in the band centered at 220 nm is bigger for the native protein than for Ol-GOx (Figure S4 in the Supporting Information), which can be attributed to the protein–protein association. This thermal stabilization (based on the decrease in TM for Ol-GOx) is an important advantage in the synthesized Ol-GOx oligomeric form. In this case, the unfolding appears to be irreversible.32 The CD results were corroborated using vibrational spectroscopy; the latter gave more details regarding the molecular orientation. It is important to note that Fourier transform infrared spectroscopy (FTIR) revealed a difference in the α-helix and random coil contents, which can be related to the accuracy of the technique, as opposed to CD data, which is influenced by the presence of agglomerates.33 Figures 1e and 1f show the FTIR spectra for the native protein and Ol-GOx in the region of amide I band (17001600 cm-1).34-36 The percentage of each secondary structure determined by FTIR, which corresponds to 30% of β-sheet, 43% of α-helix, 14% of β-turn and 13% of random coil to for OlGOx, shows slight differences in the percentage content of β-sheets and β-turns for native GOx (3% and 7%, respectively), suggesting a change in the secondary structure after oligomerization. This increase in β-sheets was expected, once it has been recognized as a common structural element in protein aggregates.37 In order to understand the oligomerization process and the


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increase in β-sheets, we created a theoretical model for the Ol-GOx oligomer based on protein– protein docking.38 This model do not take into consideration carbohydrates and water molecules. Comparing the Ramachandran plots between the native GOx and the Ol-GOx (Figure S5 in the Support Information), an increase in the angles that represent the formation of β-sheets could be observed, which is in agreement with the FTIR results. Full kinetic characterization was carried out for both native GOx and Ol-GOx. For this, glucose (substrate) oxidation was monitored. Fig. 2 shows the plot of glucose concentration vs formation of H2O2, Michaelis-Menten fit for glucose biocatalysis, and Lineweaver-Burk linearization. Glucose is oxidized to gluconolactone (equation 1) and the enzyme is regenerating by transferring two electrons and two protons to O2, forming hydrogen peroxide (H2O2). Hydrogen peroxide is catalytically reduced by horseradish peroxidase (HRP), while ABTS is oxidized to ABTS+ (equation 2), which has an absorption at 420 nm. Thus, the kinetics of GOx and Ol-GOx reaction was followed by measurements based on this absorption band. The reaction at 30 ºC (in air) was evaluated monitoring the absorption at 420 nm after 30 minutes of the reaction, which was stopped by addition of 0.10 mL of 4.0 mol L-1 HCl. The reaction media consisted in 0.5 mL of 16 mmol L-1 ABTS, 0.5 mL of 1.9 U mL-1 HRP and 0.5 mL of glucose in 100 mmol L-1 citrate-sodium phosphate buffer (pH 5.2). β-D-glucose + O2 → D-glucono-1,5-lactone + H2O2

(1)

ABTS + H2O2 ⇌ ABTS+ + H2O

(2)


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Figure 2. Plot of glucose concentration vs formation of H2O2 and Michaelis-Menten fit for catalysis of glucose by (a) GOx; (b) Ol-GOx; (c) Plot of Lineweaver-Burk for GOx (red line) and Ol-GOx (blue line); (d) Zoomed region of (c). For GOx, kcat values can be obtained in a broad range of values; it is very typical for GOx from Aspergillus niger. The value obtained (135 s-1) is in agreement with the literature.39,40 We propose that the oligomeric Ol-GOx can be used as a potential biocatalyst instead of its native form (the dimeric GOx). The hydrophobicity and size of Ol-GOx confers ideal surface properties for solid-state immobilization making this novel enzyme amenable for use in the fabrication of biodevices. Carbon surfaces have been used to produce bioelectrodes for fundamental studies in biosensors and their development. In terms of physiosorption, the solid surface interacts


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preferentially with the protein by van der Waals forces. Thus, the direct electrochemistry of OlGOx can be evaluated using protein film voltammetry (PFV),41 with Ol-GOx physically adsorbed on the surface of a flexible carbon fiber electrode (FCF). From this point onwards, we will denote the carbon electrode modified with native enzyme and the oligomerized GOx as FCF-GOx and FCF-Ol-GOx, respectively. The electrode preparation and enzyme immobilization were performed as described elsewhere19,42 and the details are provided in Supporting Information. The voltammograms obtained are shown in Fig. 3a. Here, direct electron transfer (DET) occurs from the FAD and the electrode surface. The Ol-GOx molecules get efficiently adsorbed and measurements >100 voltammograms were obtained without any decrease in faradaic currents, an important PFV result in terms of FAD-dependent GOx (Figure S6 in the Supporting Information). The formal potential obtained for Ol-GOx is -0.47 V at pH 7.5, which is in agreement with the theoretical value expected for FAD inside the protein.43 The cyclic voltammogram showed a peak potential separation (∆E) of 30 mV, which indicates a highly reversible process and a fast charge exchange.44 The electrochemical reaction involves 2 electrons for electro-oxidation of FADH2 to FAD, and the linear increase in faradaic currents with the increasing scan rate indicated a charge transfer-limited process (Figure S7 in the Supporting Information). For Ol-GOx, this increase was faster than the native protein. The maximum rate constant (kmax) for heterogeneous electron transfer for GOx and Ol-GOx is 380 s-1 and 2630 s-1, respectively. Thus, the charge transfer was enhanced 7-fold when Ol-GOx was used in the modified electrode. This result can be explained in terms of the Marcus theory45 (equation 3), when the reorganization energies (λ) obtained was 0.28 eV for native GOx and 0.43 eV for Ol-GOx (Figure S8 in the Supporting Information).


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The λ is a parameter that describes the energetics of nuclear motion in the redox molecule and the solvent shell surrounding the molecule, resulting from the change in the charge/medium interaction upon the spatial shift of charges.46-48 The rearrangement of charges promoted by the two electrons transferred per monomer in Ol-GOx, and the size of the protein significantly influences the solvent environment resulting in increased λ.

/ =

'

& , 1 [ [! ±#$% % ()* ] /] 23 1 - ./0 ⁄

(3)

Where kmax is the maximum electron-transfer rate constant at high overpotential, λ is the reorganization energy in eV, R is universal constant of gas, T is temperature, F is Faraday constant, and (E – Eº′) is the overpotential.


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a)

b)

c)

0.4

j / mA cm-2

0.0

-0.2

0.00 -0.40 -0.80

-0.6

-0.5

-0.4

-0.3

E / V (Ag/AgClsat)

d)

-0.80

Ar O2

-1.20 -0.4

0.00

-0.40

-0.4 0.0 0.4 0.8

-0.4 0.0 0.4 0.8

E / V (Ag/AgClsat)

E / V (Ag/AgClsat)

e)

f)

0.60

0.60

Ol-GOx

0.40

j / mA cm-2

GOx

0.24 V

0.20

0.40

0.15 V

0.20

0.00

0.00

-0.20 0.2

0.3

0.4

0.5

0.6

E / V (Ag/AgClsat)

Ar O2 O2 + glucose

-1.20

O2 + glucose

0.40

j at 0.45 V / mA cm-2

j / mA cm-2

0.2

0.40 Ol-GOx

GOx

j / mA cm-2

0.40

j / mA cm-2

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-0.20 0.1 0.2 0.3 0.4 0.5 0.6

0.35 0.30 0.25 0.20

E / V (Ag/AgClsat)

0

4

Cglucose

8

12

mmol L-1)

Figure 3. DET and bioelectrocatalysis of GOx and Ol-GOx. (a) Cyclic voltammograms of FCFGOx (red line) and FCF-Ol-GOx (blue line) bioelectrodes. Scan rate: 100 mV s-1. Electrolyte support: 0.1 mol L-1 sodium phosphate buffer (pH 7.5). Temperature 25 °C. Argon atmosphere; Inside: cyclic voltammograms after the subtraction of the capacitive current. Cyclic voltammograms of (b) FCF-GOx and (c) FCF-Ol-GOx in absence of oxygen (black line), in presence of oxygen (red line), and in presence of oxygen after the addition of 13.3 mmol L-1 of glucose in the electrolyte (blue line); Zoomed region of catalytic response of (d) FCF-GOx bioelectrode and (e) FCF-Ol-GOx bioelectrode, both in presence of oxygen, in absence of glucose (black line), and after successive additions of glucose. Final concentration of glucose: 13.3 mmol L-1. Scan rate: 50 mV s-1. Electrolyte support: 0.1 mol L-1 sodium phosphate buffer (pH 7.5). Temperature 25 ºC; (f) Plot of glucose concentration vs current density at 0.45 V for GOx (●) and Ol-GOx (●), a description of the statistical treatment of error analysis.

Finally, we show the bioelectrocatalysis of glucose by using FCF-GOx and FCF-Ol-GOx electrode (Figs. 3d, e, and f). In the presence of molecular oxygen, glucose is converted to gluconic acid with concomitant oxygen reduction to hydrogen peroxide.49,50 The consumption of


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oxygen is related to the irreversible process at -0.3 V, as shown in Fig. 3. This process is associated with the hydrogen peroxide production and its oxidation occurs at 0.24 V for GOx and 0.15 V for Ol-GOx. The maximum current for glucose oxidation at 0.45 V enhanced by about 30 %, as shown in the quasi steady-state region in the plot (Fig. 3f). The catalytic activity of the enzyme in the FCF-GOx and FCF-Ol-GOx bioelectrodes was studied by chronoamperometry (Fig. 4a and 4d). For enzyme electrodes, the applied potential was +0.70 V instead of +0.45 V because it ensures that the current-time transient reach steady-state plateau, which is necessary to obtain the kinetic parameters. The glucose solution (0.9 mol L-1) was prepared and stored overnight. For all experiments, oxygen gas was purged for 10 minutes prior to the measurements. In order to obtain KMapp, it was utilized Lineweaver-Burk (Fig. 4b and 4e) plots and HanesWoolf plots (Fig. 4c and 4f). For Lineweaver-Burk approach, equation 4 was utilized to gives an indication of the enzyme-substrate kinetics, which is the electrochemical version of the Lineweaver-Burk equation.51,52 0

455

=

0

4

+

99

78

0

(4)

4 :

Where ISS is the steady-state current after the addition of substrate, Imax is the maximum current measured under saturated substrate conditions, and c is the bulk concentration of substrate. Consequently, utilizing this approach, the KMapp for GOx is 11 mmol L-1 and jmax is 233 μA cm-2, while for Ol-GOx these values correspond to 4.2 mmol L-1 and 445 μA cm-2, respectively. For Hanes-Woolf plot53, equation 5 could be used to give us an indication of the same parameters described above, considering that jmax is proportional to vmax, which provides the same values of these parameters for GOx and Ol-GOx that those obtained by LineweaverBurk approach.


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