Laccase-Mediator System Using A Natural Mediator As A Whitening Agent For The Decolorization Of Melanin Part 1
Apr 27, 2023
Abstract: In this study, a laccase-mediator system (LMS) using a natural mediator was developed as a whitening agent for melanin decolorization. Seven natural mediators were used to replace synthetic mediators and successfully overcome the low redox potential of laccase and limited access of melanin to the active site of laccase. The melanin decolorization activity of laccases from Trametes versicolor (lacT) and Myceliophthora thermophila (lacM) was significantly enhanced using natural mediators including acetosyringone, syringaldehyde, and acetovanillone, which showed low cytotoxicity. The methoxy and ketone groups of natural mediators play an important role in melanin decolorization. The specificity constants of lacT and lacM for melanin decolorization were enhanced by 247 and 334, respectively, when acetosyringone was used as a mediator. LMS using lace and acetosyringone could also decolorize the melanin present in the cellulose hydrogel film, which mimics the skin condition. Furthermore, LMS could decolorize not only synthetic eumelanin analogs prepared by the oxidation of tyrosine but also natural melanin produced by melanoma cells.
According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
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david.deng@wecistanche.com WhatApp:86 13632399501
1. Introduction
Laccases (EC 1.10.3.2, benzenediol: dioxygen oxidoreductases) are multicopper proteins that catalyze the oxidation of various phenolic and non-phenolic compounds via a radical-catalyzed reaction mechanism by the reduction of molecular oxygen [1,2]. Laccases have been used as biocatalysts for biodegradation processes, such as the bioremediation of dyes [3,4], pharmaceuticals [5,6], herbicides [7], and delignification [8–10]. Laccases have also been used to catalyze the polymerization of dye precursors and organic compounds [11]. In particular, their attractive properties, such as low substrate specificity, the use of oxygen as the final electron acceptor, the generation of water as a by-product, and no demand (or no production) of peroxides, make them interesting in biotechnological and environmental fields [1,11,12].
Four copper ions at the active site are involved in the catalytic activity of laccase. “Blue” copper (T1 site) oxidizes the substrate, and the trinuclear copper cluster (T2/T3) receives the electrons from the T1 site to reduce the molecular oxygen [1,12,13]. In particular, the redox potential of the T1 site Cu is considered a major factor in determining the catalytic ability of laccases [14]. Laccases possess a relatively low redox potential (0.4–0.8 V) compared to ligninolytic peroxidases (over 1 V) such as manganese peroxidase and lignin peroxidase. Laccases cannot directly oxidize non-phenolic substrates with a redox potential above 1.3 V [13,14]. Therefore, to overcome the limitations of laccase, laccase-mediator systems (LMS) using small molecular compounds, such as 2,20 -azinobis(3- ethylbenzthiazoline-6-sulphonate) (ABTS), 1-hydroxy benzotriazole (HOBt), violuric acid (VLA), N-hydroxy phthalimide (HPI), N-hydroxy acetanilide (NHA), and TEMPO, which act as redox mediators, have been suggested [15–17].
These mediators permit the oxidation of bulky compounds via different oxidation routes. The laccase-ABTS system oxidizes substrates by generating a cationic ABTS radical via an electron transfer (ET) mechanism. LMSs with HOBt, VLA, HPI, or NHA produce nitroxyl radicals via the hydrogen atom transfer (HAT) mechanism [1,12,17]. Furthermore, mediators such as TEMPO and its analogs react via ionic pathways to generate oxoammonium ions [1,12,18]. The use of these mediators can oxidize a wide range of compounds in various applications, such as dye degradation [3,4], drug degradation [5,6], and lignin degradation [8–10]. Nevertheless, the applications of synthetic mediators in industrial fields have been limited due to their potential toxicity, high cost, and enzyme inactivation effect. Recently, lignin-derived phenolic molecules as natural mediators (e.g., syringaldehyde, acetosyringone, vanillin, acetovanillone, methyl vanillate, ferulic acid, sinapic acid, p-coumaric acid, etc.) have been studied to replace synthetic mediators [1,12]. The advantages of natural mediators are low cost and low toxicity because they are obtained from natural and renewable sources [19].
Melanin is a group of natural pigments produced by melanogenesis through the oxidative polymerization of tyrosine by melanocytes. Natural melanin can be classified into five categories of eumelanin, pheomelanin, all melanin, pheomelanin, and neuromelanin [20]. Recently, various medical and electrochemical applications using melanin or melanin precursors have been studied [20,21]. Human skin color is mostly determined by the presence of melanin. In the cosmetic industry, the direct depigmentation of melanin using enzymes has been proposed for the development of skin-whitening agents. Several peroxidases have been studied to decolorize melanin. Woo et al. showed that synthetic melanin can be directly decolorized by lignin peroxidase from P. chrysosporium [22]. The Keneko and Mohorˇciˇc groups also reported the enzymatic decolorization of melanin by manganese peroxidase isolated from fungi (Sporotrichum pruinose and Phlebia radiata) [23,24]. Kim et al. reported that crude enzyme mixtures containing manganese peroxidase, lignin peroxidase, and laccase showed melanin depigmentation activity [25]. When peroxidases decolorize melanin, they require hydrogen peroxide (H2O2) as a cofactor that can irritate the skin. Thus, to reduce the usage of H2O2, glucose oxidase or laccase was introduced into the enzyme combination system [26,27]. Laccases can decolorize melanin without the use of hydrogen peroxide. Khammuang and Sarnthima reported that laccase from Lentinus polycarpous Lév showed melanin decolorization activity using ABTS, vanillin, and vanillic acid as mediators [28].
2. Materials and Methods
2.1. Materials
2.2. Melanin Decolorization by LMS
The saturated melanin solution (1.4 mg/mL) was prepared by the dissolution of 3 mg synthetic melanin in 1.3 mL of 10 mM NaOH. The solution was centrifuged at 8500 rpm for 5 min to remove the undissolved melanin, and the supernatant was diluted with 0.1 M citric acid phosphate buffer (pH 3, 4, 5, 5.5, 6, or 7) and used as a substrate solution for LMS. The concentration of melanin in the substrate solution was 63 µg/mL and spectrophotometrically confirmed at 475 nm. The 0.8 mL of melanin substrate solution was mixed with 0.1 mL mediator solution (0–1 mM) in a 1.5 mL Eppendorf tube. The melanin decolorization reaction was initiated by adding 0.1 mL of laccase solution (15.8 µg (0.6 U) lacT or 19.2 µg (1.8 U) lacM) to a mixture of melanin and the mediator at 25 ◦C in a shaking water bath at 120 rpm. After the reaction, the reaction mixture was centrifuged, and the absorbance of the supernatant was measured at 475 nm. The decolorization yield (%) was calculated using the following equation:
Decolorization (%) = (A0 − At)/A0 × 100, (1)
2.3. Kinetic Study of Melanin Decolorization by LMS
2.4. Cytotoxicity of Natural Mediators
The B16F10 melanoma cell line (Korea Cell Line Bank, Seoul, Korea) was used to determine the cytotoxicity of natural mediators for LMS. A neutral red (NR) assay was performed to measure the cytotoxicity of the mediators [29]. NR measures the viability of live cell lysosomes. Melanoma cells with a concentration of 3 × 104 cells were dispensed into each well of a 96-well plate. After 24 h of cultivation, the cells were treated with natural mediators (1, 2, 5, 10, 22, and 46 mM). After additional cultivation for 2 days, the cells were treated with 50 µg/mL NR solution dissolved in DMEM and incubated for 3 h. After removing the supernatant through suction, an NR desorb solution (1% glacial acetic acid, 49% ethanol, and 50% distilled water) was used for color extraction. After the extraction process, the change in absorbance was measured at 540 nm.
2.5. Preparation and Decolorization of the Melanin/Cellulose Hydrogel Film
To measure the decolorization activity of LMS for the melanin/cellulose film, the prepared hydrogel film was cut into a 1 × 2 cm sheet. The hydrogel film was immersed in 4 mL of 0.1 M citric acid phosphate buffer (pH 5.5); subsequently, 0.5 mL of 1 mM acetosyringone and 0.5 mL of lace solution (2.5 U) were added to the buffer. The de-colorization reaction was carried out in a water bath with shaking at 120 pm and 25 ◦C for 3 h. After the reaction, the film was washed with distilled water and attached to the inner side of the cuvette to measure the change in the spectra in the range of 400–800 nm using a UV/Vis spectrophotometer. Control reactions without lacM or mediators were also conducted under the same conditions. The release of the melanin from the film or color change of the melanin/cellulose film was not detected under the reaction conditions. Furthermore, the change in color parameters (L*, a*, and b* values) of the melanin/cellulose film after the decolorization reaction by LMS was also recorded using a colorimeter (KONICA MINOLTA, Tokyo, Japan). The ∆L (metric lightness difference), ∆E (total color difference), YI (yellowness index), and WI (whiteness index) values were obtained using the following equations [30–32]:
2.6. Preparation of Natural Melanin
Natural melanin was obtained from B16F10 melanoma cells. The cells were treated with alpha-melanocyte-stimulating hormone to produce melanin. After 4 days of incubation, the cells were captured using trypsin-EDTA and sonicated for 10 min. The supernatant was obtained by centrifugation at 8000 rpm for 10 min and then adjusted to pH 1.5 using 6 M HCl. The solution was boiled at 100 ◦C for 4 h to hydrolyze the residual protein fractions. The solution containing natural melanin was washed with acetone, followed by chloroform and ethanol, and then washed with deionized water to eliminate residues, such as cells, media components, and protein fractions [33,34]. All washing processes were performed more than twice. Finally, natural melanin was obtained by freeze-drying and used as a substrate for LMS.
3. Results and Discussion
3.1. The Effect of Mediators on Melanin Decolorization by LMS
The effect of various mediators on the melanin decolorization reaction by LMS was investigated using two laccases from T. versicolor (lacT) and M. thermophila (lacM) (Figure 1). When lacT was used without a mediator for melanin decolorization, the decolorization yield was only 1% after 5 h of reaction. When HOBt was used as a mediator for lacT, the decolorization yield was slightly enhanced to 2% after 5 h of reaction. The use of various synthetic mediators, such as HOBt, ABTS, VLA, and TEMPO, in the laccase-catalyzed oxidation of phenolic or non-phenolic compounds significantly enhanced the reaction rates [10,15]. When the access of target compounds into the active site of laccase is limited by their steric hindrance, mediator radicals formed by laccase can efficiently oxidize the target compounds by the electron transfer or hydrogen atom transfer mechanism [12]. HOBt is one of the most commonly used synthetic mediators in LMS due to its high redox potential (1.1 V) [6]. However, HOBt is not a good cosmetic ingredient because of its potential cell toxicity and ability to inactivate laccase. Thus, we selected seven natural mediators, acetosyringone, syringaldehyde, p-coumaric acid, vanillin, vanillic acid, vanillyl alcohol, and acetovanillone, for the melanin decolorization reaction by LMS. Interestingly, all of the natural mediators act as more efficient mediators than HOBt for melanin decolorization by lack. When acetosyringone, syringaldehyde, and p-coumaric acid were used, the decolorization yields were 28%, 22%, and 18%, respectively, after 5 h of reaction. These results demonstrate the usefulness of natural mediators for the melanin decolorization reaction by LMS. The mediators in the LMS are oxidized to mediator radicals by laccase, and the mediator radicals induce the oxidation and decolorization of melanin. When lack was used without a mediator for melanin decolorization during a sufficient reaction time, which could reach the equilibrium state, the decolorization yield was 7% after 24 h of reaction. The natural mediators, except vanillic acid, act as more efficient mediators than HOBt for melanin decolorization by lacT after 24 h of reaction. The decolorization yield after 24 h reaction using vanillic acid as a mediator was lower than that after 5 h of reaction. This may be caused by the low stability of the oxidized radical form of vanillic acid. When acetosyringone, syringaldehyde, and acetovanillone were used, the decolorization yields were 34%, 30%, and 31%, respectively, after a 24 h reaction. p-Coumaric acid was more efficient in enhancing the initial reaction rate than acetovanillone, while acetovanillone induced a higher decolorization yield at the equilibrium state than p-coumaric acid.
The effect of the mediator on the decolorization reaction by LMS using lacM was also very similar to that obtained by LMS using lacT. When lace was used without a mediator for melanin decolorization, the decolorization yield was only 2% after 5 h of reaction. HOBt as a mediator for lacM did not enhance the decolorization yield during the 5 h reaction. All of the natural mediators, except p-coumaric acid and vanillin, acted as efficient mediators of melanin decolorization by lacM. When acetosyringone and syringaldehyde were used, the decolorization yields were 25% and 22%, respectively, after 5 h of reaction. p-Coumaric acid and vanillin were used as efficient mediators for each, but they could not efficiently enhance the decolorization rate in LMS using lace. This may be caused by the lower substrate specificity of lacM for p-coumaric acid and vanillin. The decolorization yields after 24 h of reaction of lacM with p-coumaric acid and vanillin were similar to those obtained by lacT. This indicates that the oxidized forms of p-coumaric acid and vanillin can efficiently decolorize melanin, although their oxidation rate by lacM was much lower than that by lacT. When lacM without a mediator was used for melanin decolorization during a sufficient reaction time, the decolorization yield was 5% after 24 h of reaction. The natural mediators, except vanillic acid, also act as more efficient mediators than HOBt for melanin decolorization by lacM after 24 h of reaction. When acetosyringone, syringaldehyde, and acetovanillone were used as mediators for lacM, the decolorization yields were 34%, 28%, and 31%, respectively, after 24 h of reaction. When vanillic acid was used as a mediator for both lacT and lacM, it showed the lowest decolorization yield. This may be caused by the low stability of the oxidized radical form of vanillic acid. Khammuang and Sarnthima reported that vanillin and vanillic acid could be used as mediators for melanin decolorization using laccase from Lentinus polycarpous [28]. However, they showed much lower decolorization activity for melanin than acetosyringone when they were used as mediators for lacT and lace.
These results indicate that natural mediators are more efficient for melanin decolorization by LMS than HOBt. HOBt has been considered as an efficient synthetic mediator for laccase because of its high redox potential and the catalytic role of the NOH group of HOBt [5]. The efficiency of mediators to oxidize target substrates is highly dependent on the ability to form stable radicals as well as the steric hindrance caused by bulky alkyl substituents rather than the redox potential of the mediators [19,35]. The low stability of the oxidized intermediate of HOBt has been determined through cyclic voltammetry [6]. Therefore, the low decolorization yield by LMS using HOBt may be caused by the low stability of HOBt under the reaction conditions of laccase. Although the redox potential of syringaldehyde was lower than that of HOBt, syringaldehyde showed relatively higher stability than HOBt [6].
As shown in Figure 2, the natural mediators used in this work have various substituents (e.g., hydroxyl, methoxy, carboxyl, ketone, or aldehyde) at different positions on the benzene ring [12,19]. Mediators with two methoxy groups (acetosyringone and syringaldehyde) showed higher decolorization rates than those with one methoxy group. The decolorization rate obtained by p-coumaric acid with no methoxy group was dependent on the type of laccase. The p-coumaric acid with lacT showed a higher decolorization rate than those with one methoxy group, while p-coumaric acid with lacM showed the lowest decolorization rate in the 5 h reaction. Fillat et al. also showed similar results for the decolorization of flexographic inks by fungal laccases with natural mediators [36]. The phenolic natural mediators (acetosyringone, methyl syringe, and syringaldehyde) with two methoxy substituents in the ring were oxidized by laccase faster than p-coumaric acid with no methoxy group. This indicates that methoxy groups play a more important role as electron donors than the double bond of the lateral chain of p-coumaric acid. When the mediators with one methoxy group were compared, the decolorization yield increased in the following order: acetovanillone > vanillin > vanillyl alcohol > vanillic acid. Acetovanillone, which has a ketone group, showed a higher decolorization rate and yield than the mediators with aldehyde, hydroxyl, and carboxyl groups. Acetosyringone with a ketone group also showed a higher decolorization rate and yield than syringaldehyde with an aldehyde group.
In the following experiments, acetosyringone, syringaldehyde, and acetovanillone, which showed high melanin decolorization ability, were selected as mediators for LMS to decolorize melanin. The effect of the mediator on the decolorization reaction by LMS was investigated over time (Figure S1). The decolorization reaction using lacT with acetosyringone, syringaldehyde, and acetovanillone resulted in 21%, 18%, and 1% decolorization yields after 1 h of reaction, respectively. The decolorization reaction using lacM with acetosyringone and syringaldehyde resulted in 19% and 18% decolorization yields after 1 h of reaction, respectively. Both laccases showed similar reaction profiles when the same mediator was used. Acetosyringone and syringaldehyde significantly enhanced the decolorization rate during the initial reaction. These results show that acetosyringone and syringaldehyde containing dimethoxy groups were more efficient in enhancing the initial rate of decolorization by LMS than acetovanillone containing one methoxy group. Fillat et al. also reported that the methoxy groups in the ring structures of mediators act as accelerators for the oxidation of substrates [36]. On the other hand, the decolorization yield after 24 h of reaction by acetovanillone was similar to that by syringaldehyde, although acetovanillone moderately enhanced the reaction rate.
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