Lignin peroxidase is a promising ingredient in skin whitening cosmetics because it is able to oxidize melanin with a high redox potential. Crude lignin peroxidase from fungal fermentation is commonly used due to difficulties in expression and purification. Lignin peroxidase is difficult to express and purify, so it has usually been used as a crude form for cosmetics.
The efficiency of melanin decolorization was reached to 73% with the intermittent addition of hydrogen peroxide (H2O2), since the excess concentration of H2O2 converts LiPH8 to compound III, which is the deactivated form of lignin peroxidase. Considering that the intermittent supply of H2O2 is not practical for cosmetic applications, glucose oxidase from aspergillus niger (GOx) was used for the on-site generation of H2O2.
Introduction
Thus, the inactivation of lignin peroxidase by H2O2 is the main obstacle to achieving high melanin decolorization efficiency. In this work, in order to obtain an enzyme with high purity, the lignin peroxidase isoenzyme H8 (LiPH8) was expressed in Escherichia coli. Since GOx can continuously generate low-concentration H2O2 from molecular oxygen and β-D-glucose (glucose), a significant increase in melanin decolorization efficiency was expected.
The redox mediator, the veratryl alcohol cation radical, decolorizes eumelanin and turns it into PTCA redox mediator, the veratryl alcohol cation radical decolorizes eumelanin and turns it into PTCA (pyrrole-2,3,5-tricarboxylic acid), which is a possible candidate for the oxide form of melanin.
Results and discussion
The intermittent addition of H2O2 was performed to support the assumption that the melanin decolorization efficiency could be improved by keeping H2O2 concentration low and continuing to supply H2O2. As a result, melanin decolorization efficiency was achieved up to 73%, which is more than twice as high as 31.9% in the previous experiment. However, melanin decolorization was inhibited in the case of 1400 μM H2O2 with 12 minute time interval, even the instantaneous concentration of H2O2 did not exceed 280 μM (Fig. 3).
However, adding more enzyme will not improve the melanin decolorization efficiency as it is saturated with about 1000 μM H2O2 and approaches 73%. In addition, addition of H2O2 at an instantaneous concentration lower than 300 μM did not inhibit melanin decolorization by LiPH8. This result confirmed that melanin decolorization can be effectively ameliorated by intermittent addition of H2O2 at time intervals that prevents inactivation of LiPH8.
However, it is not possible to apply H2O2 repeatedly on the skin in the case of cosmetic applications.
Conclusion
Materials and Methods
- Expression and purification of recombinant lignin peroxidase H8
- Lignin peroxidase activity assay for VA
- Melanin decolorization catalyzed by LiPH8
- Melanin decolorization catalyzed by LiPH8 with intermittent addition of H 2 O 2
- Melanin decolorization catalyzed by LiPH8 with in-situ generated H 2 O 2 by GOx - 20
Without exception, all melanin decolorization experiments were performed with 50 mg/L synthetic melanin at 100 rpm with magnetic bar stirring at room temperature. Ionic strength was controlled by KCl concentration, and the effect of ionic strength ranging from 0 mM to 1000 mM on melanin decolorization was also investigated. The reaction was initiated by the addition of H2O2 in a reaction mixture containing BR buffer, LiPH8, VA and synthetic melanin with a final volume of 2 mL.
The melanin decolorization efficiency was estimated from the absorbance difference between reaction solution and control solution. The melanin destaining was performed in pH 4.0 BR buffer with the above conditions except for H 2 O 2 . Destaining of melanin was performed in BR buffer (pH 4.0), 300 mM β-D-glucose with glucose oxidase ranging from 0 U/mL to 0.24 U/mL instead of adding H2O2.
Definition of one unit of GOx is the amount of enzyme that catalyzes glucose and produces 1 μmol of H2O2 per minute. Glucose concentrations from 0 mM to 300 mM were used to analyze the effect of glucose varying concentrations on decolorization of melanin with the above condition for 1 hour. The control experiment was performed with identical condition as mentioned above without adding glucose oxidase.
Control experiments for all other substances were also performed to understand the effect of each reaction component such as GOx, glucose, LiPH8, VA and H2O2. Inactivating effect of phenolic unit structures on lignin biodegradation by lignin peroxidase from Phanerochaete chrysosporium. A randomized, placebo-controlled trial to compare the skin lightening efficacy and safety of lignin peroxidase cream vs.
Effects of hydroquinone and its glucoside derivatives on melanogenesis and antioxidation: biosafety as skin whitening agents. Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium. Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger.
In silico-engineered lignin peroxidase from Phanerochaete chrysosporium exhibits improved acid stability for lignin depolymerization. Recombinant lignin peroxidase-catalyzed melanin decolorization using in situ generated H2O2 for use in whitening cosmetics.
Outline of melanin decolorization catalyzed by lignin peroxidase and glucose oxidase
Melanin and redox mediator must interact directly or the melanin decolorization will be reduced since the VA cation radical has a very short lifetime [33, 34]. The melanin decolorization efficiency was significantly reduced at pH 5.0 and pH 6.0 while it reached more than 30% at pH 4.0 in 1 h as the optimum pH of LiPH8 for VA oxidation was around pH 3.0. It is clear that high specific activity of LiPH8 for VA is essential for higher decolorization efficiency (Fig. 1).
Effect of pH on melanin decolorization efficiency (bar graph, left axis) and specific activity of LiPH8 for VA (line graph, right axis). Since H2O2 acts as the final electron acceptor in melanin decolorization with LiPH8, more H2O2 is required for better melanin decolorization. However, lignin peroxidase is sensitive to inactivation in high concentration of H2O2 by the formation of compound III or heme degradation [21].
Melanin decolorization efficiency reached 31.9% with 250 μM H2O2 and then decreased with increasing concentration of 250 μM H2O2 (Fig. 2). These results imply that additional H2O2 is required for higher melanin decolorization efficiency, but should keep the concentration of H2O2 low. Therefore, this study made an effort to supply low concentration of H2O2 continuously, which can improve the overall melanin decolorization efficiency.
Effect of H2O2 concentrations melanin decolorization efficiency (bar graph, left axis) and specific activity of LiPH8 for VA (line graph, right axis). Melanin decolorization was performed with 0.06 U/mL LiPH8, 50 mg/L synthetic melanin and 2 mM veratryl alcohol in BR buffer pH 4.0 for 1 hour. Specific activity of LiPH8 for veratril alcohol was measured with 0.02 μM LiPH8 and 2 mM veratril alcohol in pH 3.0 condition.
The reaction was performed with 0.06 U/mL LiPH8, 50 mg/L synthetic melanin, and 2 mM veratril alcohol at pH 4.0. Reactions were performed with 0.06 U/mL LiPH8, 300 mM glucose 50 mg/L synthetic melanin, and 2 mM veratryl alcohol under pH 4.0 condition. Total H2O2 concentration up to 1400 μM was divided and added at 12 min, 15 min, 20 min, 30 min and 60 min intervals over 1 h to support that the instantaneous high H2O2 concentration is the main factor of LiPH8 inhibition .
