Synthesis of glucose oxidase-PEG aldehyde conjugates and improvement of enzymatic stability

Abstract

In this article, aldehyde derivative of poly(ethylene glycol) (PEG) was synthesized directly with sodium periodate agent. To obtain a conjugate which possesses better stability, PEG aldehyde was bonded to native enzyme with different molar ratios. The conjugation reaction turned out to be efficient and mild. Colorimetric method was applied to evaluate the enzymatic activity of native GOD and its derivatives by introducing another enzyme, horseradish peroxidase. The GOD–PEG aldehyde conjugate with polymeric chains exhibited reduced enzymatic activity towards the catalytical oxidation of glucose, but with significantly increased thermal stability and elongated lifetime. When GOD was modified with PEG aldehyde the enzymatic activity was decreased 40% at 30 °C. However, when incubated at 60 °C the GOD–PEG aldehyde conjugate still retained the enzyme bioactivity of 40% bioactivity left after 4 h, whereas the native GOD lost almost all the activity in 4 h. The polymer chain attached, the more reduction of the enzymatic activity resulted, however, the longer the lifetime and higher thermal stability of the enzyme obtained.

Keywords:

• Thermal stability
• covalent conjugation
• glucose oxidase
• PEG aldehyde
• activity

Introduction

Enzymes are highly efficient catalysts widely employed in biotechnology due to their excellent biological activities and extraordinary selectivity, as well as environmentally friendly properties. Enzymes are extremely expensive because of their difficulties in obtaining and purifying. Denaturation is simply changes from the native structure, without alteration of amino acid sequence and without severance of any of primary chemical bonds [1–5]. Therefore, the industrial use of such expensive biocatalysts suffers from a critical point, which is the lack of efficient recovery process. Immobilization of enzymes can improve the catalytic activity, selectivity and thermal stability [6–14].

To create a stable and affordable immobilization method, such as non-covalent interactions or covalent binding of biological material have been developed [9,10,15–17]. Bioconjugates with polymers have a high probability of scientific research and application. Modification of functional polymers with biomolecules can improve properties, such as stability, solubility and biocompatibility. For example, protein conjugation with poly(ethylene glycol) (PEG) has been shown to significantly increase blood circulation times in vivo. PEGylation can also increase the protein stability in vivo by reducing susceptibility to proteolytic enzymes and antibodies. In recent years, advanced research has been done on this area and great advances in medicine and biotechnology have been made achieved [18–23].

PEG is a water-soluble polymer with excellent properties, such as minimum interface free energies with water, extensive hydration, good structuring flexibility, chain mobility and a large excluded volume [24,25]. Thus, PEG would be used as an outstanding flexible chain to connect enzyme, which could improve the rigidity and stability of the enzyme. PEG has excellent solubility in both water and organic solvents. These materials exhibit no antigenicity, immunogenicity or toxicity and are consumed in human body as approved by the U.S. American Food and Drug Administration [26–28].

Glucose oxidase (GOD, EC 1.1.3.4) is a flavoprotein that catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone using molecular oxygen as the electron acceptor and releases hydrogen peroxide [29–31]. GOD is widely applied in food industries in many countries. With the principle of substrate specificity, GOD could be used in manufacturing glucose biosensors for quick, accurate, and easy determination of the glucose level in food, beverage, blood, and so on [23].

GOD is a easily broken enzyme; therefore, its modification needs quite mild condition. That is probably the reason why the manipulation of the bioactivity of GOD through surface modification was rarely reported. Herein, we report the synthesis of a biocompatible polymer, PEG aldehyde derivative for covalent conjugation to GOD to obtain enzyme–polymer conjugates towards the manipulation of the enzyme’s bioactivity, thermal, and storage stabilities.

Experimental

Reagents

GOD from Aspergillus niger (Mw = 186 kDa) (FL.49180) and o-dianisidine (FL.33430) were purchased from Fluka. D-(+)-Glucose (G 7528) and PEG (Mw: 20 kDa, FL.95172) were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were analytical grade.

Preparation of PEG aldehyde derivatives

Freshly prepared NaIO₄ solution (8 g dissolved in 70 ml distilled water) was added slowly over PEG solution (3.33 g dissolved in 30 ml distilled water) and kept stirred in darkness for 24 h at room temperature. After this period, the solution was dialysed against distilled water for 24 h and aldehyde derivative of PEG was recovered by freeze drying [10,32].

Preparation of GOD–PEG aldehyde conjugates

Amount of PEG aldehyde derivatives needed for the preparation of conjugates were calculated according to the formula given below for constant enzyme concentration (0.2 mg/ml).

n_GOD/n_PEG = c_GODM_PEG/c_PEGM_GOD = 1/1; 1/5; 1/10; 1/20

where c shows concentration (mg/ml) while M is molar weight.

PEG aldehyde and enzyme solutions were freshly prepared in phosphate-buffered saline (PBS) buffer at pH 7.0. Subsequently, the reaction was started by mixing enzyme (2 ml), PEG aldehyde (2 ml) solutions and incubating in a water bath at 25 °C for 16 h. As a result, a schiff base was formed between aldehyde groups of PEG derivative and amine groups of enzyme. Later, 5.6 ml cold (+4 °C) 100 mM sodium bicarbonate solution at pH 8.5 was added in order to stop the reaction. To reduce the schiff base formed and unreacted aldehyde groups of PEG derivative, 9.6 mg of sodium borohydride was added. This solution was stirred for 15 min and then again, 9.6 mg of sodium borohydride was added to their action medium. Reduction reaction was continued at +4 °C for 15 min and pH of the final solution was adjusted to 7.0 [10].

Activity determination

The test tubes containing 780 μl PBS buffer were incubated in a stirring waterbath at working temperatures for 5 min. The reaction was iniated at working temperatures (30, 40, 50, 60, 70 °C) by adding 50 μl glucose (%25 w/v), 25 μl o-dianisidine (10 mM), 15 μl HRP (0.005 mg/ml), and 30 μl GOD solution (0.0025 mg/ml), respectively, for the initiation of this reaction. After 10 min, reaction was stopped by adding 100 μl 1M H₂SO₄ solution. The total volume was completed to 1 ml [33,34]. Subsequently, UV-visible spectra of these reaction products and their A₄₀₀ values were recorded. Activities in units were calculated according to the formula given below.

U/mg = A₄₀₀ × 10⁶/ɛtc_GOD

ɛ: molar absorption coefficient of o-dianisidine at 400 nm (17.500) t: incubation time (minutes) c_GOD: GOD concentration (mg/ml) A₄₀₀: absorbance at 400 nm

Stabilities of the conjugates and native GOD were determined at different and pH values (5, 6, 7, 8) according to the same procedure for activity determination described above.

Characterization of the conjugate

The molecular masses of PEG aldehyde, free enzyme and conjugate were estimated by HPLC, using column Shim-Pack Diol-300 (7.9 mm ID × 50 cm) with Shim-Pack Precolumn Diol (4.0 mm ID × 5 cm) at room temperature. The fractions were evaluated at 1 ml.min⁻¹ with 0.1 M PBS buffer (pH 7) containing 0.15 M NaCl and 7.5 mM NaN₃. The evaluate was monitored at 280 nm with Shimadzu SPD-10AV VP Model UV-vis Dedector. The particle size of the enzyme and conjugate were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with 4.0 mV He-Ne laser at a wavelength 633 nm at a temperature 25 °C. All the solutions were filtered through 0.2 μm RC-membrane filters (Sartorius) before DLS measurement.

Kinetic studies

To measure kinetic parameters, substrate (o-dianisidine) concentrations from 0.25 to 2.5% (W7V) were used as using constant enzyme concentration under the optimum conditions (at pH 5.0, 25 °C). Rates were measured for five different substrate concentrations in duplicate. The apparent kinetic constants Michaelis–Menten constants (K_m), V_max and K_cat of native GOD and GOD–PEG aldehyde conjugates (n_GOD/n_PEG 1:5) were estimated by using linear regression plots of Lineweaver and Burk.

Kinetics of thermal inactivation at 60 °C

Native enzyme and n_GOD/n_PEG 1:1 and n_GOD/n_PEG 1:5 conjugates were incubated at 60 °C in 50 mM sodium acetate buffer, pH 5.0. Aliquots were removed at scheduled times (60, 120, 180, 240 min), chilled quickly, and assayed for enzymatic activity.

Determination of storage stability

Sample preparations containing 0.0025 mg/ml native GOD and GOD–PEG aldehyde conjugates (n_GOD/n_PEG 1:1 and n_GOD/n_PEG 1:5) were incubated in acetate buffer (pH 5.0) at 4 °C. Enzyme activity was assayed at proper time intervals in order to determine the remaining activity of the enzyme. All the experiments were carried out in triplicate and the results represent mean values with less than 2% of error.

Scanning probe microscope (SPM) analysis

Surface morphologies of native and dextran-treated GOD were observed with scanning probe microscope SPM 9600 (Shimadzu, Japan) in dynamic mode using a Silicon cantilever. The force constant and resonance frequency of the cantilever used was 42 N/m and 320 kHz, respectively. Scanning rate and resolution was set to 1 Hz and 256 × 256 pixels, respectively. Samples were prepared by depositing a drop of commercial GOD- or PEG-treated GOD on freshly cleaved mica surface. Sample surface was rinsed with ultra pure water and dried at room temperature after 5 min of incubation. Each SPM image was obtained using an area of 0.5 μm × 0.5 μm.

Results and discussion

The modification of GOD by covalent conjugation with PEG aldehyde using different molar ratios (n_GOD/n_PEG 1/1; 1/5; 1/10; 1/20) was studied. The peak of aldehyde observed at about 2936 cm⁻¹ of Fourier transform infrared (FTIR) spectra was disappeared when covalent bondings occurred between aldehyde groups of PEG derivatives and primer amino groups of enzyme molecules (results were not submitted). The stabilities of conjugates were evaluated with the activities determined at different temperatures and pH values.

One of the most fundamental parameters for characterizing macromolecules is their molecular weight and molecular weight distribution [35]. However, gel permeating chromatography has been widely used technique for estimating these characteristics of proteins, polysaccharides, protein–polymer conjugates in their native forms based on their elution positions [36–38]. HPLC method is used for the determination of soluble aggregates. Figure 1 shows the HPLC results of GOD–PEG aldehyde conjugate (n_GOD/n_PEG 1:20), native enzyme and aldehyde derivative of PEG at pH 7.0. Evaluation position of the conjugate which was placed in front of GOD and aldehyde derivative of PEG, relates the formation of covalently bonded macromolecules with higher molecular weight than the constituents. This result is in agreement with the results reported in case of enzymes with dextran polymer [5,38].

The fluorescence prosthetic group flavin adenine dinucleotide (FAD) indicates different spectral characteristics in different proteins. Each subunit of dimeric GOD contains one tightly bound FAD as cofactor and 10 tryptophan residues. The energy transfer probability from tryptophan residues to the flavin cofactor could be affected from both distance and orientation due to structural perturbation of this enzyme [5,39–41]. In this work, FAD fluorescence intensity of native GOD and the conjugate (n_GOD/n_PEG 1:20) was measured for 120 min at 60 °C; pH 4, 5, 6, 7 and 8 (Figure 2). these values are in agreement with our previous studies reported in case of GOD with dextran [5].

Figure 1. HPLC chromatograms of GOD–PEG aldehyde conjugate (n_GOD/n_PEG 1:20) and free GOD.

Figure 2. Changes in FAD fluorescence intensities of native GOD and a GOD–PEG aldehyde conjugate (n_GOD/n_PEG 1:5) depending on time at 60 °C for different pH values, λ_ex= 400 nm, λ_em= 500 nm; pH 4 (A), pH 5 (B), pH 6 (C), pH 7 (D), pH 8 (E).

Kinetics of thermal inactivation

Thermal denaturation of GOD generally results in the release of the co-substrate, FAD from the GOD, thus causing the GOD deprived of its bioactivity [23].

Figure 3 reports the kinetics of thermal inactivation of the enzyme forms exposed at 60 °C. All GOD preparations lost activity progressively with time according to a biphasic inactivation kinetics, but the GOD–PEG aldehyde conjugates (n_GOD/n_PEG 1:1 and n_GOD/n_PEG 1:5) were more resistant to thermal treatment than the native counterpart. From Figure 3, we can see that both the two kinds of conjugate exhibited the similar trend: the residual activity declined along with the reaction time prolonged while the n_GOD/n_PEG 1:1 declined less and more slowly compared with n_GOD/n_PEG 1:5. After 240 min, native GOD lost the activity while conjugates did not lose activity until the reaction time was at 240 min.

Figure 3. Thermal inactivation of the native and the conjugates (n_GOD/n_PEG 1:1; n_GOD/n_PEG 1:5) which incubated at 60 °C for different time.

When we compare these activity results with our previous studies [42], these values are in agreement with the values reported in case of GOD with dextran.

Bhatti et al. [43] also reported that the optimal catalytic activity for GOD was observed at pH 5.8 and temperature of 45 °C. However, when GOD was dissolved in PBS (pH 5.8) and incubated at 60 °C for 10 h, no obvious bioactivity decrease was observed neither. This event might be due to the presence of K⁺ cations in the buffer as observed by Gouda et al. [44]. The presence of K⁺ cations could enhance the thermal stability by primarily strengthening the hydrophobic interactions and making the enzyme a more compact dimeric structure. We found that the native GOD lost 55% of its activity in 120 min and lost almost all the activity in 4 h. However, the GOD–PEG aldehyde conjugates only lost 10% of its activity in 120 min at 60 °C. After 4 h incubation, GOD–PEG aldehyde conjugates still retained the enzyme bioactivity of 40% bioactivity left (Figure 3). In conclusion, although the conjugates exhibited low activity than the native one, the thermal stability of modified GOD can be significantly increased.

Effect of pH value and temperature on the enzymatic activity

The effects of pH value and temperature on the activities of native enzyme and GOD–PEG aldehyde conjugates are given in Figures 4 and 5. Activities of native enzyme and conjugates against different pH values are shown in Figure 4. The optimal pH of enzyme and conjugates is 5.0. Activities of native enzyme and all conjugates showed showed enhanced stability and broader pH scope.

Figure 4. Activities of native GOD and GOD–PEG aldehyde conjugate with different molar ratios (n_GOD/n_PEG 1/1, 1/5, 1/10, 1/20) depending on pH at 30 °C.

Figure 5. Thermal stabilities of GOD–PEG aldehyde conjugates with different molar ratios (n_GOD/n_PEG 1/1, 1/5, 1/10, 1/20) at pH 5.

The operating temperature range is another important factor in limiting the practical applications of GOD. As shown in Figure 5, activity decline of native enzyme was very fast with the increase in temperature. Whereas conjugates had higher resistance against the temperature. High resistance against the temperature may be because of the formation of thick polymer cover around enzyme molecules [45]. It was observed that conjugates had slightly less activity than native enzyme, but their values were not changed significantly before 50 °C.

Enzyme kinetic data

To investigate the mechanism of enzymatic conversion, a kinetic model has been used to fit the experimental data. The catalytic parameters of native GOD and modified GOD with PEG aldehyde (n_GOD/n_PEG: 1/5) with respect to substrate were determined on the basis of typical Michaelis–Menten behaviour. The kinetic constants, Michaelis–Menten constant (K_m), maximum decolorization rate (V_max) and catalytic constant (K_cat) of native and GOD–PEG aldehyde conjugate were determined (Table 1).

Table 1. The kinetic parameters of the oxidation of glucose by GOD at pH 5.0.

Parameter	Native GOD	GOD–PEG aldehyde
Km (mM min^−1)	12.47 ± 0.09	8.92 ± 0.1
Vm (mM)	0.013 ± 0.001	0.01 ± 0.0
Kcat (min^−1)	5.13 ± 0.3	4 ± 0.01
Kcat/Km (1 mg^−1 min^−1)	0.411 ± 0.02	0.448 ± 0.03

These results are in agreement with the SPM images (Figure 6). Figure 6 shows the SPM images of the surface morphologies of the free GOD and GOD–PEG aldehyde. These SPM images clearly show the variations in the morphology of the GOD before and after PEG aldehyde conjugation. Although all specimens prepared in the same conditions, the differences observed in SPM images indicated the interaction between GOD and PEG aldehyde, hence enzyme microenvironment modification by conjugation was revealed.

Figure 6. SPM images of the free GOD (A) and modified GOD with PEG aldehyde (nE/nD:1/5) (B).

Determination of storage stability

Ying et al. [46] also searched the storage stability of GOD immobilized on micro porous membranes prepared from poly(vinylidene fluoride) with grafted poly(acrylic acid) side chains. They found that the native and the immobilized GOD stored in the phosphate buffer (pH 7.4) at 4 °C remained 35 and 55% of their initial activity after 60 days, further evidencing the elongation of the lifetime of the modified GOD. The storage stability of GOD and GOD modified with poly(PEG acrylate) was investigated by Luo et al. [23]. They found that modified GOD stored at room temperature was more stable than that without modification stored at the same temperatures. Native GOD lost its all bioactivity at in 29 days while the modified GOD still retained bioactivity of 17.3 U/mg [23].

As shown in Figure 7, GOD–PEG aldehyde conjugate (n_GOD/n_PEG: 1:1) is more stable than native GOD when stored in the acetate buffer (pH 5) at 4 °C temperature. There was an sharply and a regular decrease in the activity of native enzyme after 15 days while the activity of the conjugate was constant for nearly 25 days. After 25 days incubated, GOD–PEG aldehyde conjugate still remained 55% of its bioactivity.

Figure 7. Storage stabilities of native and modified GOD with PEG aldehyde storage at 4 °C temperature.

Conclusions

In summary, GOD was modified with covalent bondings occurred between aldehyde groups of PEG derivatives and primer amino groups of enzyme molecules. In this method, it is possible to provide a shell around the enzyme surface so that the enzyme is more resistant to environmental conditions. Chosen polymer should be reactive towards the groups located on the enzyme surface. The application of covalent conjugation of GOD improved the thermal stability and inactivation profile of enzyme activity. These findings will all help with further studies in the future application of enzyme in industrial processes such as biomedicine, food industry and biotechnology.

Disclosure statement

No potential conflict of interest was reported by the authors.