The Kinetic and Analytical Aspects of Enzyme Competitive Inhibition: Sensing of Tyrosinase Inhibitors (original) (raw)
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Biosensing of tyrosinase inhibitors in nonaqueous solvents
Electroanalysis, 1995
An amperometric biosensor based on a Clark oxygen electrode coupled with a biocatalytic layer containing tyrosinase has been developed for measurement in nonaqueous solvents. Glass filter paper was soaked with the enzyme and fixed on top of the electrode by a dialysis membrane. Thiourea derivatives were detected in hexane by evaluating the decrease in the response towards phenol. This method was fast and highly sensitive with detection limits in the range of 13-181 nM depending on the structure of the inhibitor compounds. The response time was up to 10min and depended on the inhibitor's structure. The mechanism of inhibition is discussed using kinetic parameters.
Amperometric study of the inhibitory effect of carboxylic acids on tyrosinase
Journal of Molecular Catalysis B-enzymatic, 2004
A tyrosinase-modified Pt electrode, based on physical entrapment of the enzyme in agar-agar gel, was constructed and used to investigate the inhibitory effect of six carboxylic acids. At an applied potential of −50 mV versus saturated calomel electrode (SCE), the bioelectrode develops a fast, steady state response, linearly correlated with the phenol concentration up to 10 mg/l, with a sensitivity of 3.7 nA l/mg. A kinetic analysis of the amperometric response to phenol, recorded in the absence and in the presence of carboxylic acids (benzoic, 3-bromobenzoic, 4-ethylbenzoic, acetic, phenylacetic, 2-naphthylacetic acids), revealed that for the first four compounds the inhibition process corresponds to an uncompetitive one. Using the Lineweaver-Burk linearization the inhibition constants as well as the inhibition coefficients were calculated for the strong inhibitors: benzoic, 3-bromobenzoic, 4-ethylbenzoic and acetic acids.
Biosensors and Bioelectronics, 2002
A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite-Teflon; reticulated vitreous carbon (RVC)-epoxy resin; and graphite-ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite-EPD electrodes, whereas a longer useful lifetime was observed for the graphite-Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite-EPD \ graphite-Teflon RVCepoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.
Improved mediated tyrosinase amperometric enzyme electrodes
Journal of Electroanalytical Chemistry, 1997
Tyrosinase amperometric enzyme electrodes in a carbon paste configuration based on the electroreduction of quinoid enzymatic products have been constructed. The electroreduction was mediated by the inclusion in the carbon paste material of an osmium (4,4'-dimethyl 2,2'-bip~/ridine)2(l, 10-phenanthroline-5,6-dione) mediator. The mediated electrodes showed a three-fold increase in current density, almost two orders of magnitude decrease in detection limit, and a more than one order of magnitude increase in lifetime of the sensor under FIA conditions compared with the unmediated electrodes. Furthermore, they showed a clear mass transport limited response, changing the response limiting step compared with unmediated electrodes. These findings suggest that the use of suitable mediators and electrode configurations can improve the characteristics of tyrosinase electrodes.
Screen-printed tyrosinase-containing electrodes for the biosensing of enzyme inhibitors
Talanta, 1996
Disposable amperometric inhibition biosensors have been microfabricated by screen printing a tyrosinase-containing carbon ink. The decrease in the substrate (catechol) steady-state current, caused by the addition of various pesticides and herbicides. offers convenient quantitation of micromolar levels of these pollutants. Unlike esterasebased disposable strips. the tyrosinase thick-film devices can be fabricated by incorporating the enzyme within the carbon ink. and do not require a prolonged incubation step in the presence of the inhibitor. The effect of experimental variables, such as the enzyme loading or substrate concentration. is assessed. Applicability to an untreated river water sample is illustrated. Such use of single-use devices for monitoring toxins addresses the problem of irreversible enzyme inhibition. and holds great promise for on-site field analysis.
Preventing inhibition of tyrosinase with modified electrodes
Analytica Chimica Acta, 2006
Wines, especially red wines, contain numerous biologically active compounds, the most important of which are polyphenols, whose nutritional importance is attributed to their antioxidant power. Because of this, the detection of the amount of phenolic compounds in red wines becomes extremely important. However, using free enzyme in the determination of phenolic compounds in wines cannot reflect the actual values since there are also naturally found inhibitors in red wines. In this study, benzoic acid, cinnamic acid, and sorbic acid were utilized to understand the behavior of immobilized polyphenol oxidase in the conducting polymer matrices toward inhibition. Cinnamic acid was found to be the most powerful inhibitor for both free and immobilized enzyme in copolymer matrix of poly(terephthalic acid bis-(2-thiophen-3-yl-ethyl) ester) (PTATE) with polypyrrole (PPy). In the case of immobilized enzyme in PPy matrix, it was observed that sorbic acid is a stronger inhibitor than cinnamic acid. The inhibitory effects of these inhibitors on PPO were compared with respect to both the structural differences of inhibitors and conducting polymer matrices.
Development of a paper-type tyrosinase biosensor for detection of phenolic compounds
Biotechnology and Applied Biochemistry, 2014
Phenolic compounds are the chemicals which are used by many different industries and as a result of this spread to the environment. These compounds can be absorbed easily through the human and animal skin and through the mucosal membrane, mix in to the blood circulation and thus create a toxic effect on several tissue and organs including, liver, lung and kidneys. For this reason, determination of phenolic compounds emitted to environment is a very important issue. In fact, there are standard methods for the determination of these compounds like HPLC, Spectrophotometric and calorimetric methods however, these are time consuming methods and requires to be expertise. On the other hand, there are also different types of biosensors developed for the phenolic compound detection. In this study, a new, disposable, cheap and convenient tyrosinase biosensor was developed for the phenolic compound detection. By means of absorption method, the enzyme tyrosinase and the chromophore MBTH were immobilized on the support material and as a model substrate L-dopa was used. As a result of optimization studies 1mg/ml tyrosinase concentration and 1.5mM MBTH concentration were determined for using in biosensor construction. v Detection limit of l-dopa, model substrate, found as 0,064 mM and for other phenolic compounds, 4-chlorophenol, catechol, m-cresol and p-cresol, detection limit was obtained 0.032 mM, 0.032 mM, 0.128 mM, 0.128 mM, respectively. In addition, we found that the biosensor response was not affected by pH changes ranging from 3 to 11. The stability of biosensor which is one of the important parameter for commercialization was not change through 70 days at room temperature and 4°C when compared to at the beginning response.
Sensors and Actuators B: Chemical, 2008
In this present work, a highly reversible and sensitive amperometric biosensor, based on the immobilization of tyrosinase (Tyro) by calcium carbonate nano-materials (nano-CaCO 3), was applied for determination of food preservative, benzoic acid. The detection of benzoic acid was performed via its inhibiting action on the Tyro/nano-CaCO 3 modified glassy carbon electrode. The effects of enzyme substrate type and substrate concentration on the inhibitory were investigated in detail. A potential value of −0.20 V versus SCE, and a constant catechol concentration of 6 M were selective to carry out the amperometric inhibition measurement. The inhibitor biosensor had a fast response to benzoic acid (<5 s) with a wide linear range of 5.6 × 10 −7 to 9.2 × 10 −5 M and a high sensitivity of 1061.4 ± 13 mA M −1 cm −2. The inhibiting action of benzoic acid on the Tyro/nano-CaCO 3 electrode was highly reversible (100%) and of the typical competitive type, with an apparent inhibition constant of 17 M. This inhibitor biosensor was successfully applied for the determination of benzoic acid in some real beverage sample, such as Coca-Cola, Pepsi-Cola, Sprite and Yoghurt. Results were compared to those obtained using high performance liquid chromatography, showing a good agreement.
Journal of Pharmaceutical and Biomedical Analysis, 2013
A method for creating an immobilized capillary tyrosinase (TRS) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. Tyrosinase was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide (HDB). Then, HDB solution with the same plug length as the TRS was injected again into the capillary to cover the immobilized enzyme by forming HDB-TRS-HDB sandwich-like structure. Then, the substrate of l-tyrosine was introduced into the capillary and on-line enzyme inhibition study was performed by capillary electrophoresis (CE). The enzyme activity was determined by the quantification of peak area of the product of l-DOPA. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The immobilized enzyme could withstand 25 consecutive assays by only losing 12% activity. A known TRS inhibitor, kojic acid was employed as a model compound for the validation of the inhibitor screening method. Finally, screening 19 natural extracts of traditional Chinese drugs was demonstrated. The results indicated that inhibition activity could be straightforwardly identified with the system.