Immobilization Of Polyphenol Oxidase Research Articles

Immobilization of polyphenol oxidase on chitosan/organic rectorite composites for phenolic compounds removal.

Chitosan/organic rectorite (CTS/OREC) composites were prepared and characterized by Fourier transform infrared spectrometry and X-ray diffraction. Polyphenol oxidase (PPO) was immobilized on CTS/OREC by physical adsorption (APPO) and covalent binding (CPPO). Taguchi method was applied in the optimization of immobilization conditions resulting in the highest enzyme activity of 16.37 × 103 and 8.92 × 103U/g for APPO and CPPO, respectively. APPO enzyme activity was higher than that of CPPO, while CPPO showed the higher enzyme loading capacity than that of APPO. The removal percentage of phenolic compound, including phenol (PH), 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP), by immobilized PPO was also explored. The results indicated that APPO was more efficient in phenolic compounds removal than CPPO. APPO contributed to a quick removal in the first hour, and the removal percentage of PH, 4-CP and 2,4-DCP could reach 69.3 ± 4.2%, 89.8 ± 2.5% and 93.8 ± 1.7% within 2 h, respectively. The order of removal percentage of phenolic compounds for both immobilized PPO was 2,4-DCP > 4-CP > PH. After 10 consecutive operations, the removal percentage of 2,4-DCP reached 73.2 ± 2.6% and 60.3 ± 1.5% for APPO and CPPO, respectively. The results introduced a novel support for PPO immobilization, and the immobilized PPO had great potential in wastewater treatment.

Design and synthesis of reduced graphene oxide based supramolecular scaffold: A benign microbial resistant network for enzyme immobilization and cell growth

The present study describes the synthesis, characterization and biological application of reduced graphene oxide – chitosan (GC) based benign supramolecular scaffold (SMS). Various spectroscopic and microscopic analyses established the supramolecular interaction in between rGO and chitosan. The active performance of the developed material towards microbial resistivity, in vitro cell growth and as a scaffold for enzyme immobilizing matrix illustrates its unique implementation. Immobilization of polyphenol oxidase (PPO) onto GC lowers the Michaelis- Menten constant (Km) value and facilitates to achieve maximum velocity at low substrate concentration. Importantly GC shows no noteworthy cytotoxicity towards Wistar rat macrophage cells. Moreover, incorporation of gold nanoparticle further strengthens the microbial resistance properties of GC as well as improves its biocompatibility by reducing cytotoxicity. Therefore these unique features may inspire it to appear in large scale for industrial utilization.

Development And Validation Of Biosensing Method For Acetaminophen Drug Monitoring

A polyphenol oxidase (PPO) was immobilized by employing magnetic nanoparticles-zinc oxide/zinc hexacyanoferrate (Fe3O4NP-ZnO/ZnHCF) hybrid film electrodeposited on the surface of Pt electrode. The surface functionalization of Fe3O4NP-ZnO/ZnHCF hybrid film was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of PPO. The biosensor exhibited optimum response within 4s at pH 7.0 and 35°C and linearity in the range 0.04 to 10000 μM for acetaminophen with a detection limit of 0.04 μM (S/N=3). Accuracy of the proposed sensor was found to be 99%. The use of Fe3O4NP/ZnO/ZnOHCF for construction of amperometric acetaminophen biosensor has resulted into relatively rapid response, higher sensitivity, broad linear range, lower detection limit, good reproducibility and long term stability of this biosensor. This sensing interface provides better avenue for the fabrication of various sensor.

A New Catechol Biosensor Immobilized Polyphenol Oxidase by Combining Electropolymerization and Cross-Linking Process

A highly stable and effective catechol biosensor was prepared by immobilizing polyphenol oxidase (PPO) into polyaniline (PANI) film by using the direct electropolymerization (one-step) process in conjunction with cross-linking with glutaraldehyde. The results of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) confirmed that the immobilization of PPO was successful. The biosensor has a fast response to catechol (10 s) with a linear range of 1.0–100 µM and a detection limit of 0.01 µM. The maximum response current (I max) and the Michaelis-Menten constant (k' m) were found to be 7.94 µA and 81.32 µM, respectively. The activation energy (E a) of the PPO catalytic reaction was 16.5 kJ/mol. The biosensor exhibited good stability by retaining 80% of its original activity when stored in a dry state at 4°C for up to 5 months.

Preparation of a Polypyrrole-Polyvinylsulphonate Composite Film Biosensor for Determination of Phenol Based on Entrapment of Polyphenol Oxidase

Abstract: In this paper, a novel amperometric phenol biosensor with immobilization of polyphenol oxidase (tyrosinase) on electrochemically polymerized polypyrrole-polyvinylsulphonate (PPy-PVS) film has been accomplished via the entrapment technique on the surface of a platinum electrode. The amperometric determination is based on the electrochemical reduction of quinon generated in the enzymatic reaction of phenol. The effects of pH and temperature were investigated and optimum parameters were found to be 8.0 and 30 °C, respectively. The linear working range of the electrode was 1.0 × 10(-7) - 5.0 × 10(-6) M. The storage stability and operation stability of the enzyme electrode were also studied.

Amperometric catechol biosensor based on polyaniline–polyphenol oxidase

A novel catechol biosensor was described based on the immobilization of polyphenol oxidase (PPO) into polyaniline (PANI), which was easily constructed by direct electropolymerization of aniline in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate (EMIES). The developed biosensor for the detection of catechol has a linear range of 1.25–150μmoldm−3. The maximum response current (Imax) and the Michaelis–Menten constant (k′m) are 0.62μA and 146μmoldm−3, respectively. The activation energy (Ea) of the PPO catalytic reaction is 31.1kJmol−1 in the B–R buffer. The biosensor shows good reproducibility (a relative standard deviation of 3.1% was obtained) and remarkable long-term stability (it retains 75% of the original activity after four months). The effects of potential and pH on the response current of the biosensor are also described.

Design of a biosensor based on 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H pyrrole

Immobilization of polyphenol oxidase (tyrosinase, E.C. 1.14.18.1) was achieved on a copolymer of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1 H-pyrrole [SNS(NO 2)] with pyrrole ([SNS(NO 2)]/PPy) via electrochemical polymerization. Two different substrates; catechol and l-tyrosine were used for the characterization of biosensor. The kinetic parameters of the biosensor, maximum reaction rate of the enzyme ( V max) and Michaelis–Menten constant ( K m) were determined for two different substrates. V max was found as 0.02 μmol/min electrode for both substrates. K m values were determined as 250 and 2 mM for catechol and l-tyrosine respectively. Calibration curves for enzyme activity versus substrate concentration were plotted between 0.05 and 0.5 M catechol and between 0.8 and 2.5 mM l-tyrosine. Optimum temperature and pH, operational and storage stabilities of immobilized enzyme were examined.

Immobilization of polyphenol oxidase on alginate–SiO2 hybrid gel: stability and preliminary applications in the removal of aqueous phenol

AbstractAn organic/inorganic hybrid gel of alginate–SiO2 (ALG–SiO2) was used to immobilize the partially purified potato polyphenol oxidase (PPO) for the treatment of phenolic wastewater. The influences of alginate concentration, quantity of both enzyme and tetramethoxysilane (TMOS) on immobilization were investigated. The Michaelis constant for immobilized PPO was determined as 14.7 mmol L−1 at 25 °C, and the highest activity of immobilized PPO was achieved at pH 7.0. The ALG–SiO2 immobilized PPO was more stable than the free PPO or ALG(alone) immobilized PPO. This study suggests that ALG–SiO2 immobilized PPO might be a potential tool for the removal of phenolic compounds from industrial wastewater. Copyright © 2008 Society of Chemical Industry

Immobilization of polyphenol oxidase onto mesoporous activated carbons – isotherm and kinetic studies

Investigations were carried out in batch modes for studying the immobilization behavior of polyphenol oxidase (PPO) on two different mesoporous activated carbon matrices, MAC400 and MAC200. The PPO was immobilized onto MAC400 and MAC200 at various enzyme activities 5 × 10 4, 10 × 10 4, 20 × 10 4, 30 × 10 4 U l −1, at pH 5–8, and at temperature ranging from 10 to 40 °C. The intensity of immobilization of PPO increased with increase in temperature and initial activities, while it decreased with increase in pH. Immobilization onto MAC400 followed the Langmuir model while Langmuir and Freundlich models could fit MAC200 data. Non-linear pseudo first order, pseudo second order and intraparticle diffusion models were evaluated to understand the mechanism of immobilization. The free and immobilized enzyme kinetic parameters ( K m and V max) were determined by Michaelis–Menten enzyme kinetics. The K m values for free enzyme, PPO immobilized in MAC400 and in MAC200 were 0.49, 0.41 and 0.65 mM, respectively. The immobilization of PPO in carbon matrices was confirmed using FT-IR spectroscopy and scanning electron microscopy.

Immobilization of polyphenol oxidase on chitosan–SiO2 gel for removal of aqueous phenol

A partially purified potato polyphenol oxidase (PPO) was immobilized in a cross-linked chitosan-SiO2 gel and used to treat phenol solutions. Under optimized conditions (formaldehyde 20 mg/ml, PPO 4 mg/ml and pH 7.0), the activity of immobilized PPO was 1370 U/g and its Km value for catechol was 12 mM at 25 degrees C. The highest activity of immobilized enzyme was at pH 7.4. Immobilization stabilized the enzyme with 73 and 58% retention of activity after 10 and 20 days, respectively, at 30 degrees C whereas most of the free enzyme was inactive after 7 days. The efficiency of removing phenol (10 mg phenol/l) by the immobilized PPO was 86%, and about 60% removal efficiency was retained after five recycles. The immobilized PPO may thus be a useful for removing phenolic compounds from industrial waste-waters.

Immobilization of polyphenol oxidase in conducting graft copolymers and determination of phenolic amount in red wines with enzyme electrodes

Polyphenol oxidase was immobilized in several conducting copolymer matrices. Three different types of poly(methyl methacrylate-co-methyl thienyl methacrylate) matrices were used to obtain copolymers. Immobilization of enzyme was carried out by the entrapment of the enzyme in conducting copolymer matrices during electrochemical polymerization of pyrrole through thiophene moieties of the polymers. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values and operational stabilities of the enzyme electrodes were investigated. Total amount of phenolic compounds in some Turkish red wines was analyzed using these electrodes.

Preparation of biosensors by immobilization of polyphenol oxidase in conducting copolymers and their use in determination of phenolic compounds in red wine

Electrochemically produced graft copolymers of thiophene capped polytetrahydofuran (TPTHF1 and TPTHF2) and pyrrole were achieved by constant potential electrolysis using sodium dodecylsulfate (SDS) as the supporting electrolyte. Characterizations were based on Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Electrical conductivities were measured by the four-probe technique. Novel biosensors for phenolic compounds were constructed by immobilizing polyphenol oxidase (PPO) into conducting copolymers prepared by electropolymerization of pyrrole with thiophene capped polytetrahydrofuran. Kinetic parameters, maximum reaction rate ( V max) and Michaelis–Menten constant ( K m) and optimum conditions regarding temperature and pH were determined for the immobilized enzyme. Operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase were used to determine the amount of phenolic compounds in two brands of Turkish red wines and found very useful owing to their high kinetic parameters and wide pH working range.

Enzymatic Biosensor Based on Carbon Paste Electrodes Modified with Gold Nanoparticles and Polyphenol Oxidase

AbstractThe results presented here demonstrate the important catalytic effect of a carbon paste electrode modified by dispersion of gold nanoparticles towards different electroactive compounds. The oxidation of hydrogen peroxide starts at potentials 400 mV less positive than at bare carbon paste, while the reduction, almost negligible at bare carbon paste, starts at 0.100 V. The influence of the size and amount of gold nanoparticles in the composite matrix on the response of the electrode is discussed. The incorporation of albumin within the carbon paste facilitates the dispersion of gold nanoparticles, improving substantially the catalytic effects. At carbon paste modified with gold nanoparticles and albumin, the peak potential separation for hydroquinone decreases from 0.385 V to 0.209 V while the reduction current increases from 16.6 to 75.2 μA. The immobilization of polyphenol oxidase within the carbon paste electrode modified with nanoparticles has allowed us to obtain a very sensitive biosensor for dopamine even in the presence of large excess of ascorbic acid.

Subnanomolar Cyanide Detection at Polyphenol Oxidase/Clay Biosensors

A novel, inexpensive, and simple amperometric biosensor based on immobilization of polyphenol oxidase (PPO) into Zn-Al layered double hydroxides, also called anionic clays, is applied for determination of cyanide. The detection of cyanide was performed via its inhibiting action on the PPO electrode. Measurement was carried out with 3,4-dihydroxyphenylacetic acid as enzyme substrate, the enzymatically generated quinoid products being electroreduced at -0.2 V. An extremely sensitive detection limit (0.1 nM) was obtained for cyanide. Enzyme immobilization into an anionic exchanger clay seems to cause an increase in cyanide inhibition effects because of anion accumulation in the clay matrix.

Immobilization of polyphenol oxidase in conducting copolymers and determination of phenolic compounds in wines with enzyme electrodes

Polyphenol oxidase (PPO) was immobilized in copolymers of thiophene functionalized menthyl monomer (MM) with pyrrole. Immobilization of enzyme was performed via entrapment in conducting copolymers during electrochemical polymerization of pyrrole. Maximum reaction rates, Michaelis–Menten constants and temperature, pH and operational stabilities of enzyme electrodes were investigated. Total amount of phenolic compounds in red wines of Turkey were analyzed by using these electrodes.

Layered double hydroxides: an attractive material for electrochemical biosensor design.

Electrochemical biosensors for phenol determination were developed based on the immobilization of polyphenol oxidase (PPO) within two different clay matrixes, one anionic (layered double hydroxide, LDH) and the other cationic (Laponite). The biosensor based on the enzyme immobilized in [Zn-Al-Cl] LDH shows greater sensitivity (7807 mA M(-1) cm(-2)) and maximum current (492 microA cm(-2)). Biosensor characteristics, such as Michaelis-Menten constant, recycling constant, activation energy, and permeability highlight the advantages of LDH matrixes to immobilize PPO. It appears that LDH provides a favorable environment to PPO activity. The best PPO/[Zn-Al-Cl] configuration was used to determine five different phenol derivatives reaching extremely sensitive detection limits (< or = 1 nM).

Flow injection spectrophotometric determination of isoproterenol using an avocado ( Persea americana) crude extract immobilized on controlled-pore silica reactor

An enzymatic reactor was constructed by the immobilization of polyphenol oxidase (PPO) from avocado ( Persea americana) crude extract in an inorganic support of controlled pore silica (CPS), after a previous step of silanization. This inorganic support has been used as an excellent carrier to immobilize this enzyme and the enzymatic reactor was used in a flow injection system for the determination of isoproterenol in pharmaceutical products. The procedure is based on the oxidation reaction of this drug with immobilized PPO and the product obtained was monitored at 492 nm. This system presented an analytical curve from 1.23×10 −4 to 7.38×10 −4 mol l −1 isoproterenol with a detection limit of 6.25×10 −5 mol l −1. Recoveries of isoproterenol between 98.5 and 103.1%, a relative standard deviation (R.S.D.) less than 1% ( n=10) and 36 determinations per h were obtained.

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g−1 bead and the retained activity of the immobilized enzyme was found to be 44%. The Km values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the Vmax values were found to be 1890 and 760 U mg−1 for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry

Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation

Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme.