Determination of Phenolic Compounds Based on the Tyrosinase‐ Single Walled Carbon Nanotubes Sensor

An amperometric sensor to phenolic compound was successfully constructed by immobilizing tyrosinase on the SWNTs modified glassy carbon (GC) electrode, which was covered with Nafion film. The sensitivity of the tyrosinase‐SWNTs sensor to phenol was 155 μA/mM. The tyrosinase‐SWNTs sensor also had good response to catechol, p‐chlorophenol and m‐cresol. Furthermore, benzoic acid could be detected based on the inhibition to tyrosinase activity.     

Highly sensitive sensors based on the immobilization of tyrosinase in chitosan

A novel tyrosinase biosensor has been developed for a subpicomolar detection of phenols, which is based on the immobilization of tyrosinase in a positively charged chitosan film on a glassy carbon electrode. It was found that chitosan cross-linked with (3-aminooryloxypropyl) dimethoxymethylsilane is beneficial for the immobilization of tyrosinase. The large microscopic surface area and porous morphology of chitosan matrix lead to high enzyme loading, and the enzyme entrapped in this matrix can retain its bioactivity and the positively charged surface of chitosan can also display a good anti-interference ability to the substances with positive charge. Hence, the resulting sensor offers a high-sensitivity (150 nA.nM(-1)) for the monitoring of phenols, and the detection limit is as low as 5.0 x 10(-11) M. Its response time is less than 2 s reaching 95% of the steady-state value. It may retain 75% of the activity for at least 70 days.     

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 μM compared to kojic acid (41.96 ± 0.78 μM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.     

Visualization of tyrosinase activity in melanoma cells by a BODIPY-based fluorescent probe

We have presented a fluorescent probe 1 that exhibits a fluorescence turn-on signal upon reaction with tyrosinase, and we show that it is readily employed for the assessment of tyrosinase activity and tyrosinase inhibitor activity in buffered aqueous solution, and further utilized for the visualization of endogenous tyrosinase activity in living melanoma cells.     

Biosensor based on a glassy carbon electrode modified with tyrosinase immmobilized on multiwalled carbon nanotubes

We describe a biosensor for phenolic compounds that is based on a glassy carbon electrode modified with tyrosinase immobilized on multiwalled carbon nanotubes (MWNTs). The MWNTs possess excellent inherent electrical conductivity which enhances the electron transfer rate and results in good electrochemical catalytic activity towards the reduction of benzoquinone produced by enzymatic reaction. The biosensor was characterized by cyclic voltammetry, and the experimental conditions were optimized. The cathodíc current is linearly related to the concentration of the phenols between 0.4???M and 10???M, and the detection limit is 0.2???M. The method was applied to the determination of phenol in water samples.

基于介孔碳的电化学酪氨酸酶生物传感器法测定水体中的苯酚及高效液相色谱法评价

采用新型的介孔碳材料作为固载酪氨酸酶的检测平台构建生物传感器,应用于水体环境中苯酚污染物的检测,并通过高效液相色谱法对电化学酪氨酸酶生物传感器法的准确性进行了评价。研究表明,介孔碳的"空间限制效应"能够防止酪氨酸酶(三维尺寸为6.5 nm×9.8 nm×5.5 nm)体外去折叠失活。基于介孔碳材料构建的电化学酪氨酸酶生物传感器在苯酚污染物检测方面显示了优良的性能,其重现性、灵敏度、稳定性、选择性以及检出限均比较令人满意。基于介孔碳的电化学酪氨酸酶生物传感器对苯酚污染物的检出限达到20 nmol/L,线性范围为0.1~10 μmol/L。采用基于介孔碳的电化学酪氨酸酶生物传感器和高效液相色谱法对实际水样品进行测定结果比对,结果表明该生物传感器方法检测结果准确、有效,适合于苯酚污染物突发污染事件的应急检测。     

Kinetic Evaluation of Aminoethylisothiourea on Mushroom Tyrosinase Activity

This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when l-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO₄ was added to the assay medium on tyrosinase activity.     

Kinetics of Ergothioneine Inhibition of Mushroom Tyrosinase

The native amino acid ergothioneine, a thiourea derivative of histidine, inhibits mushroom tyrosinase activity in a dose-dependent manner, with an IC₅₀ value of 1.025 mg/ml (4.47 mM). By contrast, histidine exhibited no inhibitory effect on mushroom tyrosinase activity. We characterized ergothioneine as a noncompetitive tyrosinase inhibitor using a Lineweaver–Burk plot of experimental kinetic data. The IC₅₀ value for ergothioneine scavenging of 2,2-diphenyl-1-picrylhydrazyl was 6.110 ± 0.305 mg/ml, much higher than the IC₅₀ for inhibition of tyrosinase activity which indicating ergothioneine on tyrosinase shows a weak correlation to its antioxidative activity. The results demonstrated that ergothioneine has a potent inhibition effect on tyrosinase enzyme activity, resulting from the presence of the sulfur substituted imidazole ring in ergothioneine.     

Comparison of tyrosinase biosensor based on carbon nanotubes with DPPH spectrophotometric assay in determination of TEAC in selected Moravian wines

Monatshefte für Chemie - Chemical Monthly - An amperometric biosensor based on carbon paste electrode coated with a thin layer of carbon nanotubes and Nafion film containing the tyrosinase...     

Design,synthesis and bioactivity of chalcones and its analogues

The Vernohia anthelmintica L.'s extract is one of the most popular Uygur medicines used for vitiligo. It is believed that the chalcone compounds of the plant play an important role in the treatment since they may activate tyrosinase and improve melanin production. In this study, twenty-one chalcones and nine analogues were synthesized in view of three different components of chalcone(A, B ring and a,b-unsaturated carbonyl). After biological evaluation of their activity on tyrosinase in cell-free systems,the result showed that most compounds(except polyhydroxy chalcones) possess activator effect on the tyrosinase, especially for 13a–15a, 20 a and 1b, which bearing a comparable activity to the positive control8-MOP. SAR of these tyrosinase activator was summed up for the first time as well. Finally, compound 13 a was found to increase melanin contents and tyrosinase activity 1.75 and 1.3 fold, respectively, compared with that of untreated murine B16 cells at the concentration of 40 mg/m L.     

Electrochemical Detection of Tyrosinase in Cell Lysates at Functionalized Nanochannels via Amplifying of Ionic Current Response

Tyrosinase is an important enzyme in the metabolism of melanin and catecholamine, as well as an important antigen in autoimmunity of vitiligo. However, it is still a challenge to develop a selective and sensitive sensing approach for monitoring of tyrosinase in cell environment. In this work, a sensing strategy based on ion current rectification induced by nanochannels was established to electrochemically detect inactive tyrosinase. Functionalized alumina nanochannels were decorated by tyramine as an electrochemical platform to selectively determine tyrosinase via chemically specific reaction between phenolic hydroxyl groups of tyramine and tyrosinase. On the other hand, Fe²⁺ was further bonded with dihydroxyl groups generated by tyrosinase in nanochannels to amplify charge changes, resulting in the enhancement on the detection sensitivity for tyrosinase. Based on this principle, the developed electrochemical sensor showed a good linearity towards tyrosinase from 2 to 50 U/mL with a detection limit down to 0.83 U/mL, which can fulfil the requirement for detection of tyrosinase in cells. Eventually, the present electrochemical sensor was successfully employed in the selective and sensitive determination of tyrosinase in cell lysates. The results suggested that the activity of tyrosinase in B16 cells was 1.7-fold higher than that of Hela cells.     

Aggregation and homogenization, surface charge and structural change, and inactivation of mushroom tyrosinase in an aqueous system by subcritical/supercritical carbon dioxide

The subcritical/supercritical carbon dioxide (SS CO(2)) has gained considerable attention in green chemistry industry for its advantage as nontoxic, nonflammable, and inexpensive. The effects of SS CO(2) treatments on aggregation and homogenization, surface charge, secondary and tertiary structure, and activity of mushroom tyrosinase in an aqueous system were investigated using a number of methods including dynamic light scattering (DLS), zeta potential measurement, circular dichroism (CD) spectropolarimeter, and spectrofluorometer. With a treatment time of 20 min, three treatment temperatures (35, 45, and 55 °C) and four pressures (5, 8, 12, and 15 MPa) had been selected. The aggregation and homogenization of the globular protein particles was induced by SS CO(2) as suggested by the particle size distribution (PSD) patterns that were closely related to the pressure and temperature. The surface charge of the tyrosinase decreased following the SS CO(2) treatments, and its variation tendency shows a favorable consistency with that of its PSD patterns. The α-helix conformation in secondary structure and fluorescence intensity reflecting tertiary structure also decreased, together with the λ(max) red-shifted with the increasing pressure. The results also indicated that SS CO(2) could enhance inactivation effect of the temperature on the tyrosinase with its lowest residual activity being about 60% under the condition of 8 MPa, 55 °C, and 20 min treatment time. The loss in the activity of the tyrosinase was correlated to its aggregation and homogenization effect induced by SS CO(2), which led to the change of surface charge as well as secondary and tertiary structure.     

Evaluation of tyrosinase inhibitory activity in Salak (<Emphasis Type="Italic">Salacca zalacca</Emphasis>) extracts using the digital image-based colorimetric method

A simple toolbox was developed for the evaluation of tyrosinase inhibitory activity in Salak (Salacca zalacca) extracts by the colorimetric measurement based on a photograph taken by a digital camera or a smartphone camera. The reaction of 3,4-dihydroxyphenylalanine (l-DOPA) and tyrosinase was employed to form the dopaquinone dye, which decreases with the increase of the tyrosinase inhibitor. Under the optimum conditions, the Salak extracts were examined for the tyrosinase inhibitory activity. The captured picture of dopaquinone dye product was analyzed by reading blue color intensity using an Adobe Photoshop CS6 program. The tyrosinase inhibition of the extracts was calculated from the blue color intensity, and expressed as %inhibition and IC₅₀ values. The obtained results from the developed method correlated well with those obtained from the microplate reader instrument. The achievement of this research will be a guideline for creating any simple analytical instrument based on colorimetry. In addition, the information on tyrosinase inhibitory activity of the Salak extracts is useful for the application of this fruit to produce the supplement food and the cosmetic in the future.     

Bioelectrocatalysis of Dopamine Using Adsorbed Tyrosinase on Single-Walled Carbon Nanotubes

Abstract

A wealth of information on the reactions of redox-active sites in proteins can be obtained by voltammetric studies in which the protein sample is arranged as a layer on a suitable electrode surface. Here, we describe a method for the performance of a tyrosinase/single-walled carbon nanotubes/glassy carbon (Tyr/SWCNTs/GC) electrode, prepared by the modification of GC electrode surface by SWCNTs and adsorption of tyrosinase on the SWCNT surfaces. SWCNTs were studied with the help of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The dimensions of SWCNTs make them ideal candidates for the adsorption of proteins. The copper-containing enzyme, tyrosinase, exhibited an electrical contact with the electrode, because of the structural alignment of the enzyme on the SWCNT surfaces. The apparent Michaelis–Menten constant (K _m) for dopamine (DA) and the stability of the enzyme electrode were estimated. This method could be suitable for applications to nanofabricated devices.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Mushroom tyrosinase inhibition activity of some chromones

Currently, aloesin is used in the cosmetic industry as a whitening agent because it inhibits tyrosinase activity. Aloesin is a C-glycosylated chromone compound isolated from aloe, and it is difficult to synthesize because of C-glycosyl moiety in the molecule. The purpose of this study is to search for a new chromone compound which is easy to synthesize and which posesses stronger tyrosinase inhibitory activity than aloesin. Fourteen chromone derivatives were synthesized and screened for their mushroom-tyrosinase inhibitory activity. 5-Methyl-7-methoxy-2-(2'-benzyl-3'-oxobutyl)chromone (15) showed the strongest activity among tested compounds. Its activity was not only stronger than aloesin, but also stronger than arbutin and kojic acid. The kinetic analysis revealed a competitive inhibition of 15 with tyrosinase for the L-tyrosine binding site.     

Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.     

Glutaraldehyde activated eggshell membrane for immobilization of tyrosinase from Amorphophallus companulatus: application in construction of electrochemical biosensor for dopamine

Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. K_m and V_max values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min⁻¹, respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 °C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 °C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at −0.19 V versus Ag/AgCl (3 M KCl). Linearity was observed within the range of 50-250 μM with a detection limit of 25 μM.     

Tyrosinase inhibitors and insecticidal materials produced by Burkholderia cepacia using squid pen as the sole carbon and nitrogen source

Reports of tyrosinase inhibitors from microorganisms are rare. A tyrosinase inhibitor- and insecticidal materials-producing bacterium, strain TKU026, was isolated from Taiwanese soil and identified as Burkholderia cepacia. Among the tested chitin-containing materials, squid pen best enhanced the production of tyrosinase inhibitors and insecticidal materials. The tyrosinase inhibitory activity (5,000 U/mL) and insecticidal activity (81 %) against Drosophila larvae was maximised after cultivation on 1 % squid pen-containing medium for 3 days. The tyrosinase inhibitory activity persisted even when the culture was treated with acidic or alkaline conditions of pH 3 or 11. The activities of both tyrosinase inhibitors and insecticide remained at 100 %, even after treatment at 100 °C for 30 min. The culture supernatant after 3 days of cultivation also showed antifungal activity against Aspergillus fumigatus and Fusarium oxysporum with maximal activities of 100 and 80 %, respectively, but no antibacterial activity against Escherichia coli was observed. The tyrosinase inhibitors were assumed to be polyphenolic compounds according to the results of chromatography.