Therefore, many studies related to the techniques of skin whitening are carried out and representative of them are blocking UV or inhibiting tyrosinase and breaking down or decolorizing melanin that has already been produced and accumulated in the skin. However, due to side effects caused by chemical reagents that inhibit tyrosinase, studies on melanin decolorization by enzymes are currently paying attention as an alternative due to less toxic and selective degrading/decolorizing melanin. Lignin peroxidase used in this study is known to have enough higher redox potential than other peroxidases to decolorize melanin.
When applied to human skin as a whitening cosmetic, the stability and activity of lignin peroxidase at pH 5.5, which is close to the normal pH of human skin, should be high. Moreover, the activity and stability of lignin peroxidase under human body temperature of about 35-37 degrees Celsius should be preserved for the actual use of whitening cosmetics. So, not only using the LiPH8 enzyme prepared in our previous report, in this study, but also various other lignin peroxidase isozymes were expressed and purified as recombinant enzyme and selected with the longest stability and the highest activity. high in the human skin environment.
Introduction
Because of these side effects caused by tyrosinase-inhibiting chemical reagents, currently, studies on melanin degradation by enzymes such as laccase, manganese-dependent peroxidase, and lignin peroxidase are attracting attention as an alternative due to melanin degrading/discoloring more less toxic and more selective. These enzymes are known to oxidize melanin and change it into structures such as PTCA [12]. Also, the decolorization efficiency of manganese-dependent peroxidase and laccase was 22%–42% and 9%–21% when lignin peroxidase showed up to 70% melanin decolorization under the same condition [ 14 ].
It is known that lignin peroxidase has a sufficiently higher redox potential than other peroxidases that can oxidize the veratryl alcohol to veratraldehyde. This property of lignin peroxidase means that the non-phenolic compounds including melanin can be easily oxidized. The basic structural unit of melanin is also represented by covalently linked indoles and it is believed that most melanin is a heterogeneous polymer consisting mainly of dihydroxy indole units that exist as a mixture of both the catechol and the quinone [15 ].
Thus, not only the decolorization but also the degradation of melanin catalyzed by lignin peroxidase can be expected due to the polycyclic structural similarity between melanin and lignin [16]. In our previously published article, the melanin decolorization efficiency catalyzed by lignin peroxidase isozyme H8 was up to 80% within 8 h at pH 4 under 25 ℃ [12]. However, if lignin peroxidase is applied to human skin as whitening agents, the stability and activity of lignin peroxidase at pH 5.5, which is close to the normal pH of human skin, should be high.
In addition, the activity and stability of lignin peroxidase under human body temperature around 35~37℃ must be maintained for the actual use of whitening cosmetics. Therefore, in this study, for the first time to our knowledge, all 10 different lignin peroxidase isozymes (PcLiP01-10) encoded by the genome of Phanerochaete chrysosporium were expressed and purified as recombinant enzyme in E. And then, in-vitro melanin decolorization as well as in vivo test using stratum corneum of 3D skin model was shown to be very active and stable lignin peroxidase under human skin environment (pH 5.5, 37.
Results and Discussion
Immunohistochemical analysis with human pigmented epidermis skin model
Melanin decolorization catalyzed by PcLiPO4 was also evaluated using a 3D reconstructed human pigmented epidermis skin model. To assess whether PcLiP04 affected 3D skin, the Fontana Masson (FM) staining assay was used to visualize melanin in 3D skin. Only PcLiP04 without H2O2 and VA was topically treated for 3 days on 3D skin, which transferred melanin to the outer layer while stimulating melanogenesis with α-MSH, an inducer of melanin synthesis.
Considering that melanin was transferred to the stratum corneum of all 3D skins by pre-induction, our data suggest that PcLiP04 may contribute to skin melanin discoloration even in the absence of mediators such as H2O2 and VA. This observation seems to contradict the result of dissolved melanin decolorization, as both H2O2 and VA were essentially necessary, as shown in Figure 14. However, when the melanin decolorization experiment was performed using a 3D skin model, it was found that higher bleaching results were obtained in the skin. 3D skin model treated with enzymes only without VA and H2O2 (Figure 13).
H2O2 is known to be generated by human skin tissue cells during respiration as a byproduct [19], which is supposed to be used for melanin decolorization catalyzed by PcLiPO4. Although it is not clear which compound can alternatively be used instead of VA, electron mediator secreted by human skin tissue can be recognized by PcLiPO4 for melanin decolorization. Considering the structural similarity of reactive residue of PcLiPO 4 , soluble state of tryptophan and phenylalanine was suggested as a candidate.
As an alternative to VA, tryptophan and phenylalanine showed comparable melanin decolorization (Figure 14), which may confirm that dissolved tryptophan or its derivative can act as an electron mediator in human skin tissue. Quantification of melanin depigmentation result using 3D skin in stratum corneum was analyzed by Image J program.
Conclusion
Experimental Materials and Methods
- Production and purification of recombinant PcLiP isozymes
- Kinetic parameters of PcLiP isozymes for VA as limiting substrate
- Thermodynamic stability determined by using DSF
- PcLiP isozymes-catalyzed melanin decolorization with intermittent addition of H 2 O 2
The refolding of PcLiP isozymes was performed in 0.1 M Tris buffer (pH 8.5) containing 1.8 mM CaCl2, 0.71 mM oxidized L-glutathione, 6 M guanidine hydrochloride, and 25 μM hemin. The refolded PcLiP isozymes were dialyzed using dialysis tubing (SnakeSkin™ Dialysis Tubing, 10K MWCO) in 100 mM sodium acetate buffer with 5 mM CaCl2 in the order of pH 6, pH 4, and finally pH 6, then stored in 10 mM sodium acetate buffer (pH 6) . PcLiP isozymes were prepared and analyzed in standard 96-well plate q-PCR system (Quantitative real-time PCR, QuantStudio™ 5), using SYPRO™ Orange dye.
The obtained intrinsic fluorescence spectra were further processed to generate plots of fluorescence intensity versus temperature. Tm values for each PcLiP isozyme were determined from the maximum of the first derivatives of these plots using Protein Thermal Shift™ v1.4 software (Applied Biosystems). A 3D reconstructed model of human pigmented epidermis (phototype VI) was obtained from Skinethic Laboratories (SkinEthic, France).
Upon arrival, the 3D skin models were transferred to a 12-well plate containing the manufacturer's maintenance medium and equilibrated overnight at 37 °C in 5 % CO2. For melanin overexpression and migration, 3D skin models were cultured with the manufacturer's growth medium containing 100 nM α-MSH (Sigma Aldrich) for 3 days. The 3D skin models were fixed in 10% neutral formalin (Sigma Aldrich) and embedded in paraffin wax.
Paraffin-embedded 3D skin was cut into 4-μm-thick sections and stained with Fontana Masson (FM) stain for melanin measurement. Lim, Y.-J., et al., Inhibitory effects of arbutin on melanin biosynthesis of α-melanocyte-stimulating hormone-induced hyperpigmentation in cultured brown guinea pig skin tissue. Tadokoro, T., et al., UV-induced DNA damage and melanin content in human skin differing by race/ethnicity.
Kim, Recombinant lignin peroxidase-catalyzed decolorization of melanin using in situ generated H2O2 for application in whitening cosmetics. Shin, S.K., et al., Effective degradation of melanin by a synergistic laccase-peroxidase enzyme complex for skin whitening and other practical applications. Kim, In silico engineered lignin peroxidase from Phanerochaete chrysosporium exhibits improved acid stability for lignin depolymerization.
