Immobilization Of Tyrosinase Research Articles

Immobilization of tyrosinase and alcohol oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

Immobilization of tyrosinase and alcohol oxidase is achieved in the copolymer of pyrrole with vinyl alcohol with thiophene side groups (PVATh- co-PPy) which is a newly synthesized conducting polymer. PVATh- co-PPy/alcohol oxidase and PVATh- co-PPy/tyrosinase electrodes are constructed by the entrapment of enzyme in conducting copolymer matrix during electrochemical copolymerization. For tyrosinase and alcohol oxidase enzymes, catechol and ethanol are used as the substrates, respectively. Kinetic parameters: maximum reaction rates ( V max) and Michaelis–Menten constants ( K m) are obtained. V max and K m are found as 2.75 μmol/(min electrode) and 18 mM, respectively, for PVATh- co-PPy/alcohol oxidase electrode and as 0.0091 μmol/(min electrode) and 40 mM, respectively, for PVATh- co-PPy/tyrosinase electrode. Maximum temperature and pH values are investigated and found that both electrodes have a wide working range with respect to both temperature and pH. Operational and storage stabilities show that although they have limited storage stabilities, the enzyme electrodes are useful with respect to operational stabilities.

Immobilization of mushroom tyrosinase on controlled pore glass: Effect of chemical modification

Mushroom tyrosinase immobilization on controlled pore glass with three different functional groups like alkylamino, carboxyl and arylamino is performed. The effect of the alkylamino-CPG arm extension on enzymatic stability and catalytic activity is also investigated. The results expressed in terms of ligand coupling, % yield and kinetic parameters, like the apparent K m app and V max app and the inherent K m inh and V max inh values demonstrate the best performance of tyrosinase immobilized on alkylammino-CPGs.

The Sonogel–Carbon materials as basis for development of enzyme biosensors for phenols and polyphenols monitoring: A detailed comparative study of three immobilization matrixes

Three amperometric biosensors based on immobilization of tyrosinase on a new Sonogel–Carbon electrode for detection of phenols and polyphenols are described. The electrode was prepared using high energy ultrasounds (HEU) directly applied to the precursors. The first biosensor was obtained by simple adsorption of the enzyme on the Sonogel–Carbon electrode. The second and the third ones, presenting sandwich configurations, were initially prepared by adsorption of the enzyme and then modification by mean of polymeric membrane such as polyethylene glycol for the second one, and the ion-exchanger Nafion in the case of the third biosensor. The optimal enzyme loading and polymer concentration, in the second layer, were found to be 285U and 0.5%, respectively. All biosensors showed optimal activity at the following conditions: pH 7, −200mV, and 0.02moll−1 phosphate buffer.The response of the biosensors toward five simple phenols derivatives and two polyphenols were investigated. It was found that the three developed tyrosinase Sonogel–Carbon based biosensors are in satisfactory competitiveness for phenolic compounds determination with other tyrosinase based biosensors reported in the literature. The detection limit, sensitivity, and the apparent Michaelis–Menten constant Kmapp for the Nafion modified biosensor were, respectively, 0.064, 0.096, and 0.03μmol, 82.5, 63.4, and 194nAμmol−1l−1, and 67.1, 54.6, and 12.1μmoll−1 for catechol, phenol, and 4-chloro-3-methylphenol. Hill coefficient values (around 1 for all cases), demonstrated that the immobilization method does not affect the nature of the enzyme and confirms the biocompatibility of the Sonogel–Carbon with the bioprobe. An exploratory application to real samples such as beers, river waters and tannery wastewaters showed the ability of the developed Nafion/tyrosinase/Sonogel–Carbon biosensor to retain its stable and reproducible response.

A Disposable, Screen-Printed Electrode for the Amperometric Determination of Azide Based on the Immobilization with Catalase or Tyrosinase

A disposable, screen-printed electrode based on the immobilization of catalase or tyrosinase was developed to construct biosensors for the amperometric determination of azide. The determination principles for azide by these two methods are based on inhibiting the enzymatic consumption of an electrode-detectable substance (hydrogen peroxide or catechol) on an enzyme-immobilized electrode. Both of these methods show a sensitive detection range and a short measuring time.

Development of an amperometric biosensor based on covalent immobilization of tyrosinase on a boron-doped diamond electrode

An amperometric enzyme electrode based on direct covalent immobilization of tyrosinase on a boron-doped diamond (BDD) electrode has been developed for the detection of phenolic compounds. Combined chemical and electrochemical modifications of the BDD film with 4-nitrobenzenediazonium tetrafluoroborate, an aminophenyl-modified BDD (AP–BDD) surface was produced, and then the tyrosinase was covalently immobilized on the BDD surface via carbodiimide coupling. The response dependences of the enzyme electrode (Tyr–AP–BDD electrode) on pH of solution, applied potential, oxygen level and phenolic compounds diffusion were studied. The Tyr–AP–BDD electrode shows a linear response range of 1–200, 1–200 and 1–250 μM and sensitivity of 232.5, 636.7 and 385.8 mA M −1 cm −2 for phenol, p-cresol and 4-chlorophenol, respectively. 90 percent of the enzyme activity of the Tyr–AP–BDD electrode is retained for 5 weeks storing in 0.1 M PBS (pH 6.5) at 4 °C.

Micro-cylinder biosensors for phenol and catechol based on layer-by-layer immobilization of tyrosinase on latex particles: Theory and experiment

Abstract Microelectrode sensors for phenol and catechol are described, based on the sequential immobilization of polystyrene sulphonate, polyallylamine, tyrosinase and polyallylamine again, onto micrometer scale latex spheres, followed by the adsorption of the spheres onto electrochemically pretreated carbon fibres. The steady state responses of the fibres are analyzed in terms of a cylindrical diffusion – kinetic model. It is deduced that the adsorbed latex particles provide a relatively open film structure, resulting in a diffusion coefficient only one order of magnitude lower than the solution value, and that at minimum 2–3% of the immobilized enzyme is catalytically active. The optimised sensors exhibit linear ranges to phenol and catechol of 7–56.5 μM and 2–19.7 μM respectively, with sensitivities of 0.15 A M−1 cm−2 and 1.72 A M−1 cm−2 respectively. The limiting factor to sensor stability is desorption of latex from the fibres.

Amperometric Flow‐Injection Determination of Phenolic Compounds Using a Biosensor with Immobilized Laccase, Peroxidase and Tyrosinase

AbstractA screen‐printed four‐electrode sensor based on immobilization of laccase (Coriolus hirsutus), peroxidase (horseradish) and tyrosinase (mushroom) in the same array was developed for monitoring of phenols. The enzymes were immobilized onto a self‐assembled monolayer (4‐mercapto‐1‐butanol) modified gold surface via covalent attachment by epichlorohydrin coupling. The experimental conditions for simultaneous operation of the three enzymes were optimized based on catechol determination. The sensors were further applied for the amperometric detection of several substituted phenolic compounds, carried out using a single line flow‐injection system. Hydrogen peroxide served as co‐substrate for peroxidase. The limits of detection for phenols in aqueous solutions were in the micromolar range, one assay was completed in less than 5 min. The preliminary studies showed that the compatibility of the above mentioned enzyme array enabled the multielectrode biosensor to be applied to real samples including industrial wastewaters and surface waters.

Immobilization of tyrosinase in chitosan film for an optical detection of phenol

An optical biosensor based on the immobilized tyrosinase enzyme in a chitosan film is described for the detection of phenol. The film was prepared by depositing a chitosan solution containing tyrosinase onto a microscope glass slide via spin coating technique. The quinone produced from the enzymatic oxidation of phenol was reacted with 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce a maroon color adduct. The color intensity of the adduct was found to increase proportionally with the increase of the substrate concentrations after 15 min exposure to the substrate. The optimum enzyme loading and chitosan concentrations were found to be 0.9 mg and 2% (w/v), respectively. The biosensor also demonstrated optimum activity at pH 6–7, optimum temperature of 45 °C and a linear response in the phenol concentration range of 2.5–70.0 μM. The response characteristics of the biosensor towards different phenolic compounds were determined. It was found that the biosensor gave a linear response in the concentration range of 2.5–50.0 μM for 4-chlorophenol, 2.5–100.0 μM for m-cresol and 12.5–400.0 μM for p-cresol. The limit of detection and the apparent Michaels–Menten constant ( K m) of the biosensor were 0.9, 1.0, 1.0, 3.0 μM and 0.038, 0.037, 0.056, 0.201 mM for 4-chlorophenol, phenol, m-cresol and p-cresol, respectively. It showed good stability for at least 2 months.

Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels

The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate ( V max) and Michaelis–Menten constant ( K m) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 °C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.

Preparation and characterization of superparamagnetic chitosan microspheres: Application as a support for the immobilization of tyrosinase

AbstractSuperparamagnetic chitosan microspheres were prepared by a water‐in‐oil suspension‐crosslinking technique. To this end, magnetite particles were dispersed in a chitosan solution in acetic acid. The dispersion was added to toluene containing Span 20 as a surfactant with stirring. Chitosan solution droplets were hardened with glutaraldehyde. The magnetic chitosan microspheres obtained were characterized with scanning electron microscopy, differential thermal analysis, and vibrational magnetometry. The microspheres had a wide size distribution, ranging from 43 ± 25 to 255 ± 55 μm, that depended on the reaction conditions. The mean particle size decreased with an increase in the concentration of Span 20 or the amount of glutaraldehyde and with the addition of NaCl. However, a major size reduction was achieved by an increase in the stirring rate. Tyrosinase was immobilized on the microspheres. The immobilized enzyme retained 70% of its activity, as determined by the capacity to degrade phenolic compounds. The immobilized tyrosinase resulted in greater stability than the free enzyme. In addition, the enzyme maintained 65% of its phenol oxidation activity after 10 cycles of reuse. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 651–657, 2005

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

AbstractMushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm−3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm−3. The results show that cinnamic carbohydrate esters of D‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry

A Mediator-Free Tyrosinase Biosensor Based on ZnO Sol-Gel Matrix

A tyrosinase biosensor was developed based on the immobilization of tyrosinase in a positively charged ZnO sol-gel matrix on a glass carbon electrode. It has been found that the ZnO sol-gel matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retained its activity to a large extent. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at −200 mV (vs. SCE) without any mediator. The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within15 s and sensitivity as high as 168 μA mmol L−1. The linear range for phenol determination was from 1.5×10−7 to 4.0×10−5 mol L−1 with a detection limit of 8.0×10−8 mol L−1. The biosensor response retained 75% of the initial response after two weeks. The performance of the developed biosensor was compared with that of biosensors based on other sol-gel matrices.

Immobilization of tyrosinase in poly(ethyleneoxide) electrodes and determination of phenolics in red wines

Polyphenol oxidase was immobilized in thiophene capped poly(ethyleneoxide)/polypyrrole (PEO/PPy) and 3-methylthienyl methacrylate-co- p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP/PPy) matrices. Immobilization of the enzyme was performed via entrapment in conducting copolymers during electrochemical polymerization of pyrrole through the thiophene moiety 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 red wines of Turkey was analyzed using these electrodes.

Immobilization of tyrosinase in polysiloxane/polypyrrole copolymer matrices

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) was achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate ( V max) and Michaelis–Menten constant ( K m) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe 2O 4 nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe 2O 4–SiO 2) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 μA/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10 −6 to 2.5 × 10 −4 M with a detection limit of 6.0 × 10 −7 M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated.

Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support

The use of immobilized and stable enzymes has immense potential in the enzymic analysis of clinical, industrial and environmental samples. But their widespread use is limited due to the high cost of their production. In the present study, an effort has been made to immobilize tyrosinase directly from ammonium sulphate precipitated proteins of the mushroom ( Agaricus bisporus) on a polyclonal antibody bound to Seralose 4B support. Polyclonal antibodies were raised in male albino rabbits by injecting commercially available mushroom tyrosinase in the presence of Freund's adjuvants. Antibodies were purified from antisera by ammonium sulphate fractionation followed by DEAE-cellulose chromatography. Two distinct bands of light and heavy chains of purified IgG, appeared on SDS-PAGE. The homogeneity of the purified IgG was further confirmed by Ouchterlony double immunodiffusion. One milliliter of cyanogen bromide-activated Seralose 4B bound 9.0 mg purified IgG and retained nearly 573 tyrosinase units. Immunoaffinity bound tyrosinase was more stable against heat and pH inactivation compared to the soluble enzyme. Immobilized enzyme exhibited no change in temperature optima between 30–35 °C whereas soluble tyrosinase has temperature optima of 35 °C. Immunoaffinity bound tyrosinase retained greater fraction of activity on both sides of temperature-optima compared to soluble enzyme. However, the broadening in pH-optima was observed from pH 5.5 to 6.0 for immobilized enzyme. Moreover, immobilized tyrosinase preparation exhibited remarkably high resistance against denaturation induced by urea and water-miscible organic solvents.

Amperometric phenol biosensor based on covalent immobilization of tyrosinase onto an electrochemically prepared novel copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) film

An amperometric biosensor has been developed for the quantitative estimation of phenols in aqueous solution. The enzyme, tyrosinase, also called as poly phenol oxidase (PPO), was covalently immobilized on a novel copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) (PAPCP) film, electrochemically prepared onto an indium-tin-oxide (ITO) coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of phenol, at fixed bias voltage of −0.2 V versus Ag/AgCl in a phosphate buffer (pH 7.2). The electrode gives a wide linear response range of 1.35 –222.3, 1.6–118.8, 1.9–257.8 μM, for phenol, catechol and p-cresol, respectively. The response time is <10 s reaching to a 95% steady state current value, and 80% of the enzyme activity is retained for 4 months.

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on NH 2 groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m 2, respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.

A novel method for the immobilization of tyrosinase to enhance stability.

A new method of tyrosinase immobilization by Fuller's-earth adsorption followed by entrapment in gelatin has been developed with 98% activity immobilization yield. An appreciable increase in operational and thermal stability was observed for FEAGE (Fuller's earth-adsorbed gelatin-entrapped) tyrosinase compared with GE (gelatin-entrapped) native enzyme. FEAGE tyrosinase could be used repeatedly after intermittent storage and retained 70% of its initial activity after eight cycles. The half-life of the GE enzyme at 40 degrees C was less than 2 h, whereas the FEAGE enzyme retained about 75% of its initial activity after 2 h. Taken together our data demonstrate clearly that the technique of immobilizing tyrosinase via adsorption followed by entrapment appears promising and is hence recommended for tyrosinase immobilization for commercial production of L-DOPA (3,4-dihydroxyphenylalanine).

Mediator-free phenol sensor based on titania sol–gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method

A novel amperometric phenol sensor was constructed by immobilizing tyrosinase in a titania sol–gel matrix. The tyrosinase entrapped sol–gel film was obtained with a vapor deposition method, which simplified the traditional sol–gel process and avoided the shrinkage and cracking of conventional sol–gel-derived glasses. This matrix provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenol could be oxidized by dissolving oxygen in presence of immobilized tyrosinase to form a detectable product, which was determined at −150 mV without any mediator. The phenol sensor exhibited a fast response (less than 5 s) and sensitivity as high as 103 μA/mM, which resulted from the porous structure and high enzyme loading of the sol–gel matrix. The linear range for phenol determination was from 1.2×10 −7 to 2.6×10 −4 M with a detection limit of 1.0×10 −7 M. The apparent Michaelis–Menten constant of the encapsulated tyrosinase was calculated to be (0.29±0.02) mM. The stability of the biosensor was also evaluated.