Covalent immobilisation of horseradish peroxidase onto poly(ethylene terephthalate)–poly(aniline) composite

Process Biochemistry 39 (2004) 883–888

Covalent immobilisation of horseradish peroxidase onto poly (ethylene terephthalate)–poly (aniline) composite Samantha Salomão Caramori, Kátia Flávia Fernandes∗ Laboratório de Qu´ımica de Prote´ınas, Departamento de Ciˆencias Fisiológicas, Instituto de Ciˆencias Biológicas, Universidade Federal de Goiás, Cx.Postal 131, 74001-970 Goiania, GO, Brazil Received 11 March 2003; received in revised form 25 April 2003; accepted 19 May 2003

Abstract An alternative route for synthesis of poly(ethylene terephthalate)–poly(aniline) composite and its use for peroxidase immobilisation is presented. The composite was synthesized by submitting polyethylene terephthalate (PET) plates to hydrazinolysis treatment, followed by polyaniline (PANI) synthesis over the PET surface. The composite was characterized by photomicrography and multiple reflectance infrared spectrometry. HRP immobilisation was very efficient, presenting 40% enzyme activity retention in the following conditions: immobilisation time of 90 min, at 4 ◦ C, pH 4.5 and enzyme concentration of 0.01 mg ml−1 . The optimum reaction pH was 7.0 and the enzyme continued to recognize its substrates after the immobilisation process. A slight difference between Km and Km.app values was observed. The immobilised HRP showed higher storage stability than free enzyme. The system PET-PANIG-HRP also showed high operational stability, maintaining 70% activity after 60 days of reutilization and storage. © 2003 Elsevier Ltd. All rights reserved. Keywords: Immobilisation; PET-PANI composite; Horseradish peroxidase; Stability; Reuse; Kinetic parameters

1. Introduction Immobilised enzymes present advantages that are well known both academically and from an industrial point of view. A particular kind of material is desirable for each application, but synthetic polymers with high flexibility and broad resistance against mechanical strains are generally suitable [1]. Peroxidases have been the subject of numerous studies including biosensor development [2], clinical diagnosis [3] and bioremediation [4]. Many synthetic and natural polymeric supports have been used in these studies, including natural beads of chitin and chitosan [5], polystyrene [6], polyaniline (PANI) [7], polyethylene terephthalate (PET) [8], and composites [9]. The main objective of enzyme immobilisation is to maximize the advantages of enzyme catalysis, which is possible using a support with low synthesis cost and high binding capacity. Other important aspect to be considered is the ∗ Corresponding author. Tel.: +55-62-521-1492; fax: +55-62-521-1190. E-mail address: [email protected] (K.F. Fernandes).

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00188-2

possibility of reaction interruption by removing the immobilised enzyme, conferring to these systems a refined control over product formation, what is not possible when enzyme is free in solution. The use of PANI for HRP immobilisation was reported recently. The system PANIG-HRP showed some improvement in the properties of HRP, such as thermal, pH and storage stability [10]. This system presented good performance in a flow injection system for hydrogen peroxide determination (unpublished data). However, the use of the PANIG-HRP system in a batchwise reactor requires filtration for reaction interruption and enzyme reuse, resulting in some losses in the reuse. In this article, we report the synthesis and characterization of an inexpensive support, a composite obtained from polymerization of PANI on PET surface. The main advantage of this material is the possibility of immediate reuse of reaction medium without losses of immobilised enzyme or polymer. This material was used for horseradish peroxidase immobilisation and the properties of this system were compared to those of the free enzyme and PANIGHRP.

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2. Materials and methods 2.1. Materials Aniline, methanol and ammonium persulphate were purchased from Merck (Merck & Co., Inc., USA). PET was obtained from plastic commercial recipients. Hydrazine hydrate, pyrogallol, catechol, guaiacol, o-dianizidine, phenol, 4-aminoanthypirine and peroxidase (EC 1.11.1.7, hydrogen donor: oxidoreductase) were purchased from Sigma-Aldrich (Germany). 2.2. Preparation and characterization of PET-PANIG composite Plates of PET (10×2 mm) were incubated in a methanol/hydrazine solution (10:1, v/v) at 40 ◦ C, for 10–40 h. Plates of PET-hydrazide were then washed in ethanol 95% (v/v) and dried in vacuum. PANI synthesis was carried following the methodology of Zhang and Li [11] with some modifications. In a typical procedure, plates of PET-hydrazide were immersed in a 0.61 mol l−1 ammonium persulphate solution for 30 min and then transferred to a 0.44 mol l−1 aniline solution for 1 h, where the polymerization took place. Plates of PET-PANI were activated with 2.5% (v/v) glutharaldehyde solution at room temperature, for 60 min. The PET-PANIG composite was washed in 0.1 mol l−1 phosphate buffer at pH 6.0 and dried at vacuum. PET-PANIG plates were characterized by photomicrography (Olympus B-201) and multiple reflectance infrared spectrometry (Hartman & Braun MB series—Michelson). 2.3. Immobilisation of HRP onto the support The PET-PANIG plates were submersed in 1.0 ml of 0.1 mol l−1 phosphate or acetate buffer containing 0.01 mg ml−1 of HRP (12 EU), at 4 ◦ C, and the mixture was maintained under orbital agitation. The time necessary for the immobilisation was tested in intervals from 30 to 240 min. The optimum pH value for the immobilisation was tested from 4.0 to 5.5 in 0.1 mol l−1 acetate buffer, and from 6.0 to 8.0 in 0.1 mol l−1 phosphate buffer.

optimum assay pH was tested for native and immobilised HRP at pH values from 4.0 to 5.5 in 0.1 mol l−1 acetate buffer, and from 6.0 to 7.0 in 0.1 mol l−1 phosphate buffer. The optimum assay temperature was determined by heating immobilised HRP and substrates in a water bath for 15 min at temperatures ranging from 30 to 50 ◦ C. 2.5. Kinetic and stability parameters The kinetic parameters Km and Km.app were determinate using H2 O2 at a fixed concentration of 5.0 mmol l−1 and using pyrogallol in a concentration ranging from 1.0 to 18.0 mmol l−1 . The activity of PET-PANIG-HRP was tested using phenol/4-aminoantipyrine, o-dianisidine, guaiacol and catechol as reduced substrates in the same concentration of pyrogallol. The operational stability was tested by the reuse of immobilised HRP after its storage in 0.1 mol l−1 phosphate buffer pH 7.0, at 4 ◦ C. Five reuse cycles of 12 days were carried out over 60 days. 3. Results and discussion 3.1. Characterization of the PET-PANIG composites Photomicrographs of PET-PANIG revealed some surface modifications that were more expressive as the time of hydrazinolysis treatment was increased. As hydrazinolysis treatment proceeded, the appearance of fissures and clefts became more intense and material obtained with 40 h of hydrazinolysis was very unstable and fragile. The degree of PANI polymerization on the surface of PET was higher in the composites in which the hydrazinolysis treatment was near 20 h. Inferior hydrazinolysis resulted in low PANI coverage, and superior times results in composites that shown spontaneous release of PANI from the plates. The infrared spectra of PET, PET after hydrazinolysis and PET-PANI composites are shown in Fig. 1. Bands

2.4. Measurement of native and immobilised enzyme activity The activities of native and immobilised HRP were measured according to Halpin et al. [12] with some modification. 0.1 ml of native HRP (12 EU) or one plate of PET-PANIG-HRP was added to 2.4 ml of an assay mixture containing 0.1 mol l−1 phosphate or acetate buffer and 12.8 mmol l−1 of pyrogallol. The reactions were started with the addition of 0.5 ml of 5.0 mmol l−1 H2 O2 solution. For native HRP, the reaction was measured at 420 nm, 1 min after H2 O2 addition. The reaction of the immobilised HRP was tested at time intervals from 1 to 60 min. The

Fig. 1. Infrared spectra of PET (A), PET-hydrazide (B) and PET-PANI (C). Conditions: hydrazynolysis time 20 h; plates 2×10 mm.

S.S. Caramori, K.F. Fernandes / Process Biochemistry 39 (2004) 883–888 Table 1 The main band assignment of the PET and PANI

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3.2. Immobilisation of HRP onto the support

Material

Chemical group

Band (cm−1 )

PET

Carbonyl CH2 of glycol Ester group C–H of aromatic ring in-the-plane C–H of aromatic ring out-of-plane

1712 1407 1235–1088 1016 871–721

PANI

Quinoid ring

1598

representing PANI replaced the band characteristics of PET in composites submitted to hydrazinolysis treatment. Table 1 shows the main band assignment of PET and PANI. Composites obtained after 18 h of hydrazinolysis were used in this work.

The time necessary for HRP immobilisation is shown in Fig. 2. There were no significant differences in the immobilised activity until 90 min. After this point, a decrease on enzyme retention was observed. Apparently, the immobilisation reaction occurred in the first 30 min. Contact with the free enzyme in the solution and those immobilised after 90 min may produce layers of adsorbed enzyme that presents lower activity than those covalently immobilised in the first 30–90 min. Another possibility was pointed out by Costa et al. [13] who proposed that increasing the contact of enzyme and support might promote multiple bonds and result in enzyme deformations, compromising the active site integrity. The highest immobilisation efficiency was observed at pH 4.5 (Fig. 3). Some authors reported pH 6.0 as the optimum

Fig. 2. Effect of time on immobilisation of HRP: temperature 4 ◦ C; enzyme concentration 0.01 mg ml−1 HRP in 0.1 mol l−1 phosphate buffer, pH 6.0; measurement of activity with pyrogallol, at room temperature.

Fig. 3. Effect of pH on immobilisation of HRP, with 0.1 mol l−1 acetate buffer (4.0–5.5) and 0.1 mol l−1 phosphate buffer (6.0–8.0); immobilisation time of 90 min and enzyme concentration 0.01 mg ml−1 HRP.

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Fig. 4. Effect of temperature on assay of native HRP and PET-PANIG-HRP. Conditions as follows: immobilisation time 90 min, enzyme concentration 0.01 mg ml−1 ; pH of the coupling medium for HRP 4.5.

for HRP immobilisation in PANIG [7] and in silica activated with glutaraldehyde [14]. The pH displacement from 6.0 to 4.5 may be attributed to the presence of reactive groups of PET or PETG, which were not recovered by PANI. This hypothesis was reinforced by the finding that immobilisation of HRP on PETG was more efficient at pH 4.5 [15]. 3.3. Measurement of enzyme activity Fig. 4 shows the results obtained in the determination of optimum temperature assay for free and immobilised HRP. Both enzymes showed similar behaviour as assay temperature was varied: they showed maximum activity near 40 ◦ C and maintained their activity at temperatures above this point (Fig. 4). The thermal stability of immobilised HRP was apparently higher than that observed for free enzyme considering the reaction time for immobilised HRP (15 min) was higher in comparison to the reaction time for free HRP (1 min). Increasing in the thermal stability was also observed with PANIG-HRP system [10]. Determination of optimum reaction pH resulted in maximum activity at 7.0 for free, PANIG-HRP and PET-PANIG-HRP (Fig. 5). Immobilised HRP was able to react with all tested substrates (Fig. 6). Using the same substrate concentration, the velocity of product appearance was different for free and immobilised HRP. This fact may be attributed to adsorption of product molecules on the surface of PET-PANIG, making difficult its detection on the bulk of reaction. This observation was also reported by other researchers that used HRP immobilised on silica [16], CM-cellulose [17], microfibres of cellulose [18] and PANI [10]. 3.4. Kinetic and stability parameters Fig. 7 presents the Michaelis–Menten and Lineweaver– Burk plots for immobilised HRP. Table 2 shows the values

Fig. 5. Optimization of pH for reaction of native and immobilised HRP. Conditions as in Fig. 4; --- native enzyme; immobilised enzyme.

obtained for Km and Km.app . There was a slight difference between the values obtained for free and immobilised enzyme, indicating the system PET-PANIG did not promote meaningful interference on HRP activity. Similar findings were reported by Fernandes et al. working with HRP immobilised onto PANI [10]. Data of operational stability showed that after five cycles of storage and reuse, the immobilised enzyme maintained 70% of its initial activity. These results represent a good performance, if compared to 5 days stability of HRP Table 2 Kinetic parameters of native and immobilised HRP determined by different methods Method

Km (mmol l−1 )

Km.app (mmol l−1 )

Lineweaver–Burk Hanes–Woolf Hyperbolic fitting

6.5 7.03 6.67

6.03 6.02 5.94

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Fig. 6. Relative reactivity of native and immobilised HRP. Conditions as in Fig. 4; absorbance of substrates tested: 420 nm pyrogallol, 510 nm phenol, 470 nm o-dianizidine, 410 nm guaiacol and 380 nm catechol.

Fig. 7. Doble-reciprocal plots of Michaelis–Menten (A), Lineweaver–Burk (B) and Hanes–Woolf (C) for immobilised HRP. Conditions as in Fig. 4; pyrogallol was utilized in concentration of 1.0–18 mmol l−1 .

immobilised by metal-ion carrier [19] and 21 days stability of HRP immobilised in CPG [20].

4. Conclusions The hydrazinolysis reaction was able to expose reactive groups on PET molecule that were partially recovered

by PANI polymer, resulting in different reactive groups for HRP bonding. The immobilisation of HRP was a viable process under optimal conditions, with 40% of enzyme retention. The support PET-PANIG did not promote meaningful changes in the microenvironment of the HRP. Thermal stability, pH profile and kinetic parameters Km.app tests supported this conclusion. Finally, the enzyme acquired operational stability after

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immobilisation, which permitted its reuse for prolonged periods of time.

Acknowledgements Samantha Salomão Caramori thanks CAPES for fellowship support.

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